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Uncovering the “riddle of femininity” in osteoarthritis: a systematic review and meta-analysis of menopausal animal models and mathematical modeling of estrogen treatment
Medical Scientist Training Program, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USACellular and Molecular Pathology Graduate Program, University of Pittsburgh, Pittsburgh, PA, USADiscovery Center for Musculoskeletal Recovery, Schoen Adams Research Institute at Spaulding, Rehabilitation Hospital, Boston, MA, USADepartment of Physical Medicine & Rehabilitation, Harvard Medical School, Boston, MA, USA
Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA, USAMcGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
Discovery Center for Musculoskeletal Recovery, Schoen Adams Research Institute at Spaulding, Rehabilitation Hospital, Boston, MA, USADepartment of Physical Medicine & Rehabilitation, Harvard Medical School, Boston, MA, USADepartment of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA, USAMcGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USADepartment of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
Post-menopausal women are disproportionately affected by osteoarthritis (OA). As such, the purpose of this study was to (1) summarize the state-of-the-science aimed at understanding the effects of menopause on OA in animal models and (2) investigate how dosage and timing of initiation of estrogen treatment affect cartilage degeneration.
Design
A systematic review identified articles studying menopausal effects on cartilage in preclinical models. A meta-analysis was performed using overlapping cartilage outcomes in conjunction with a rigor and reproducibility analysis. Ordinary differential equation models were used to determine if a relationship exists between cartilage degeneration and the timing of initiation or dosage of estrogen treatment.
Results
Thirty-eight manuscripts were eligible for inclusion. The most common menopause model used was ovariectomy (92%), and most animals were young at the time of menopause induction (86%). Most studies did not report inclusion criteria, animal monitoring, protocol registration, or data accessibility. Cartilage outcomes were worse in post-menopausal animals compared to age-matched, non-menopausal animals, as evidenced by cartilage histological scoring [0.75, 1.72], cartilage thickness [−4.96, −0.96], type II collagen [−4.87, −0.56], and c-terminal cross-linked telopeptide of type II collagen (CTX-II) [2.43, 5.79] (95% CI of Effect Size (+greater in menopause, −greater in non-menopause)). Moreover, modeling suggests that cartilage health may be improved with early initiation and higher doses of estrogen treatment.
Conclusions
To improve translatability, animal models that consider aging and natural menopause should be utilized, and more attention to rigor and reproducibility is needed. Timing of initiation and dosage may be important factors modulating therapeutic effects of estrogen on cartilage.
. Although the medical community no longer subscribes to this mentality, the consequences of these biases remain engrained within medical research and practice. From 1997 to 2000, the United States Food and Drug Administration removed 10 medications from the market; eight of these were removed because they caused adverse events in women
. Despite this incident marking the importance for studying mechanisms underlying sex-differences, single-sex preclinical studies in males still outweigh those in females 5.5 to 1.
. This disconnect between pre-clinical and clinical outcomes is likely owing to the underappreciated fact that most female rodents spontaneously rejuvenate their ovarian follicles in middle-age, and thus, do not undergo menopause
. The lack of an OA phenotype in aged female mice highlights a critical need to understand the role of menopause in OA pathogenesis.
The gap in our understanding of menopausal effects on OA may be a contributing factor to the consistent failures in developing disease-modifying treatments for OA
. The timing hypothesis suggests that HT may exert differential effects according to whether it is administered in the early or late stages of menopause
. For example, it has been shown that HT given early in menopause exerts some protective effects against sex hormone-sensitive diseases, such as cardiovascular disease
, there is a need to study this phenomenon in cartilage specifically.
Despite the high prevalence of OA in post-menopausal women and lack of therapeutic progress, the last systematic review examining menopause and OA in animal models was published in 2008
. While this work started the conversation related to OA and menopause, it only included ovariectomy models, lacked meta-analyses, did not evaluate rigor and reproducibility, and did not include multi-factorial analyses of estrogen treatment
. To expand on this pivotal work, the purpose of this study was to (1) summarize the science aimed at understanding the impact of menopause on OA in animal models and (2) investigate how dosage and timing of initiation of estrogen treatment affects therapeutic benefits on cartilage. We first performed a systematic review of the literature aimed at studying OA using menopause models in animals. Next, we performed a rigor and reproducibility analysis of these studies and subsequent meta-analyses on outcome measures of cartilage degeneration. Finally, given inconsistent clinical outcomes following HT
, we generated a series of mathematical models to evaluate the role of timing and dosage of estrogen treatment on cartilage degeneration.
Methods
Systematic review
A systematic review was performed under the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) protocol (Appendix S1)
Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group.
. Our database searches identified articles that studied the effects of menopause (defined as loss of responsiveness to ovarian hormones) on predefined articular cartilage
Impact of exercise on articular cartilage in people at risk of, or with established, knee osteoarthritis: a systematic review of randomised controlled trials.
. Studies were excluded if non-articular cartilage (e.g., fibrocartilage) were studied, but no restrictions were placed on the animal species used, joint studied, date of publication, or age of animals. For inclusion, studies must have included age-matched controls, been published in a peer-reviewed journal, and been written in English.
On August 30, 2021, a literature search was conducted on the following electronic databases: PubMed, Physiotherapy Evidence Database (PEDro), Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Cochrane Central Register of Controlled Trials (Search Terms listed in Table S1). A manual search on Google Scholar was also performed. Two independent reviewers (GG and NJ) assessed the eligibility of each article by screening titles, abstracts, and full text produced by the search based on the Cochran Handbook recommendations
. All screening steps were performed in pre-designed spreadsheets, and disagreements between reviewers were discussed until consensus was reached.
One reviewer (GG) compiled relevant study information from included articles (Table S2). When study information was unclear, the corresponding author of the study was contacted by email, and if there was no response initially, a second email was sent several weeks later. If still no response, unclear data were excluded. Data presented graphically were converted to numerical data using a digital ruler previously utilized in systematic reviews for this purpose
. Intra-rater reliability for graphical to numerical data conversion, calculated as the reviewer measuring the same data in sessions 2 months apart (n = 5), was determined to be ‘excellent’ (intraclass correlation coefficient: 0.9752, 95% CI: [0.9413, 1.0000]).
Rigor and reproducibility assessment
Two independent reviewers (GG and AB) assessed the rigor and reproducibility of each study using the ARRIVE guidelines 2.0
. Each of the 21 categories was ranked as “clearly insufficient” (0), “unclear if sufficient” (1), or “clearly sufficient” (2). Total scores ranged from 0 to 42, with a higher score indicating a more rigorous study. Disagreements between the reviewers were discussed until a consensus was reached. Spearman's correlation analysis between ARRIVE score and the year of study publication was performed using SPSS Statistics for Windows, version 28.0 (IBM Corp., Armonk, NY, USA).
Meta-analysis and other statistical analyses
Meta-analyses were performed on overlapping cartilage outcomes between studies. Standardized mean differences (SMD) and pooled standard deviations (SDpool) of outcome measures were calculated using the DerSimonian-Laird method
. All meta-analyses and other statistical analyses not related to the mathematical modeling were performed using SPSS Statistics for Windows, version 28.0 (IBM Corp., Armonk, NY, USA).
Given there is a strong effect of age (time) on cartilage degeneration, we assessed the correlation between SMD of the histological score and time after menopause that joints were collected using Spearman's correlation analysis. Upon observing there was no correlation, we statistically evaluated any differences based on confounders. Specifically, menopause induction method (estrogen receptor knock-out (ER-KO) vs overectomy (OVX)) and OA induction method (OA induced vs naturally acquired) were evaluated using Mann Whitney U tests. Species (rat vs mouse), joint (hand, knee, lumbar facet), and histological scoring method (proteoglycan depletion, erosion, Osteoarthritis Research Society International (OARSI) score, Mankin score, histological index) were evaluated using Kruskal–Wallis tests. Spearman's correlation test was used to evaluate relationships between age and sample size with SMD.
Mathematical modeling
To gain a deeper understanding of the role of estrogen treatment in mitigating OA, mathematical models were developed to simulate how cartilage degeneration, as quantified by histological score, is affected by the timing of initiation of estrogen treatment after menopause induction and estrogen dosage. Briefly, data identified in our systematic review that studied estrogen treatment effects on cartilage degeneration were input into a model (Equation (1)):
(1)
where SMD is the standardized mean difference between the estrogen-treated group vs the untreated group. SMD is defined such that zero indicates no difference in cartilage degeneration between the treated and untreated groups, positive values indicate more severe cartilage degeneration in the untreated group, and negative values indicate more severe cartilage degeneration in the treated group. The independent variable for simulations (either time after menopause induction that treatment was started or dosage) is represented by x. An ordinary differential equation (ODE) solver was used to predict the coefficient values, a and b, with the null hypothesis being that a and b are equal to zero (i.e., there is no relationship between timing/dosage and cartilage degeneration). For more information on the model, see the Supplementary Methods and Appendices S3–S5.
Results
Most studies investigating menopause-induced OA used young, ovariectomized rodents
Our systematic search identified 131 articles related to OA and menopause. After title, abstract, full text, and citation screening, 36 manuscripts were eligible (Fig. 1, Tables S2–S3)
Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice.
Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM).
Alterations to cell metabolism in connective tissues of the knee after ovariohysterectomy in a rabbit model: are there implications for the postmenopausal athlete?.
The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover.
Amelioration of collagen-induced arthritis and immune-associated bone loss through signaling via estrogen receptor alpha, and not estrogen receptor beta or G protein-coupled receptor 30.
Role of endogenous and exogenous female sex hormones in arthritis and osteoporosis development in B10.Q-ncf1∗/∗ mice with collagen-induced chronic arthritis.
Treatment with recombinant lubricin attenuates osteoarthritis by positive feedback loop between articular cartilage and subchondral bone in ovariectomized rats.
Combined effect of bilateral ovariectomy and anterior cruciate ligament transection with medial meniscectomy on the development of osteoarthritis model.
Effects of ovariectomy and estrogen therapy on type II collagen degradation and structural integrity of articular cartilage in rats: implications of the time of initiation.
Three-dimensional visualization and pathologic characteristics of cartilage and subchondral bone changes in the lumbar facet joint of an ovariectomized mouse model.
. The most common reason for exclusion was the potential for confounders (e.g., the experimental group received both an ovariectomy and OA-inducing joint injury, while the sham group received neither). Of the 36 studies, the most utilized animal models were mice (n = 17) and rats (n = 17). Most animals in the included studies were considered ‘young’ at the time of menopause induction (n = 30; Table S2). Most studies investigated OA in the knee (n = 30), with OVX being the most utilized model for menopause (n = 33).
Fig. 1Our systematic review resulted in 36articles aimed at studying menopause and osteoarthritis in preclinical models. Summary of systematic review workflow. Details on article screening steps are available in the Methods and in Table S1–S3.
. Over 50% of the included studies were “satisfactory” in study design (94.6%), experimental animals (86.5%), experimental procedures (73.0%), results (83.8%), abstract (89.2%), objectives (97.3%), ethical statement (81.1%), interpretation/scientific implications (64.8%), generalizability/translation (62.2%), and conflicts of interest (56.8%). However, the majority of included studies were “unsatisfactory” in defining inclusion and exclusion criteria (89.2%), description of animal care and monitoring (86.5%), protocol registration (97.3%), and data access statements (94.5%).
Fig. 2The literature aimed at understanding menopause and osteoarthritis in preclinical models has not become more rigorousorreproducible with more recent publications. A) The percentage of studies in each ARRIVE Guideline Category is shown. B) The relationship between year of publication and Total ARRIVE Guideline score is shown, with a higher score indicating a more rigorous study. There was no relationship between year of publication and total ARRIVE score. Table S4 contains individual ARRIVE scoring information for each of the included studies.
We also investigated whether there is a correlation between manuscript publication year and the total ARRIVE guideline score [Fig. 2(B)]. Given publication of the ARRIVE Guidelines and other rigor and reproducibility recommendations by the NIH over the last decade
, we hypothesized scores would increase over time. However, we found no correlation between ARRIVE score and year of publication, though this lack of relationship may be due to underpowered analyses. Regardless, there is clearly an ongoing need to improve rigor and reproducibility in the publication of animal studies investigating menopause and OA.
Cartilage degeneration is more severe in menopause-induced animals compared to age-matched controls
Meta-analysis on histological scoring revealed that cartilage degeneration was more severe in animals that had undergone induced-menopause when compared to age-matched controls [Fig. 3(A)]. Given the high prevalence of low methodological rigor in our review, we repeated our meta-analysis including only studies that randomized animals and used a blinded scorer for histopathological analyses. Cartilage degeneration was again more severe in animals with induced-menopause compared to the age-matched, non-menopausal controls [Fig. S1(A)]. These findings are in alignment with clinical studies showing increased incidence of hand and knee OA following the onset of menopause
and suggest menopause may have a causative role in the pathogenesis of OA. Given that the bulk of studies were performed in young animals, these findings also suggest this effect may be independent of aging.
Fig. 3In comparison to age-matched, non-menopausal controls, menopausal animals had significantly worse cartilage degeneration, as measured by histological scoring, cartilage thickness, type II collagen expression, and CTX-II. A) Meta analysis of histological score of cartilage degeneration. B) Histological score of cartilage degeneration over the course of time. C) Meta analysis of cartilage thickness. D) Meta analysis of type II collagen expression. E) Meta analysis of urinary c-terminal cross-linked telopeptide of type II collagen (CTX-II); SMD = standardized mean difference; OVX = ovariectomy; ER KO = estrogen receptor knockout.
We observed no correlation between severity of cartilage degeneration and the time following menopause induction in the included studies [Fig. 3(B)]. As this lack of relationship was unexpected, we evaluated whether confounding variables, such as the menopause model utilized, may be masking an underlying correlation. Regardless of time point, cartilage degeneration was worse in OVX animals compared to ER-KO animals (U = 72, 95% confidence interval (CI) [43, 90], P = 0.025), suggesting non-estrogen related effects of OVX may play a critical role in the development of OA. The species, joint, age, histological scoring method, sample size, and OA induction method had no effects on the observed trend (Table S5). However, it is unclear if these results are driven by underpowered analyses or if they represent a true lack of effect.
Meta-analyses on markers of cartilage health reported across studies revealed that both cartilage thickness and type II collagen were lower in menopause groups compared to non-menopause groups [Fig. 3(C, D)]. Similarly, urinary c-terminal cross-linked telopeptide of type II collagen (CTX-II), which is a biomarker for early identification of OA
Combined detection of serum CTX-II and COMP concentrations in osteoarthritis model rabbits: an effective technique for early diagnosis and estimation of disease severity.
Brief report: Cartilage thickness change as an imaging biomarker of knee osteoarthritis progression: data from the Foundation for the National Institutes of Health Osteoarthritis Biomarkers Consortium.
Relationship between CTX-II and patient characteristics, patient-reported outcome, muscle strength, and rehabilitation in patients with a focal cartilage lesion of the knee: a prospective exploratory cohort study of 48 patients.
used middle-aged animals), we do not anticipate age-related factors affected results. Taking these analyses together, these findings suggest the onset of simulated menopause contributes to cartilage degeneration.
When we performed additional meta-analyses, we observed urinary cartilage oligomeric matrix protein (COMP) was higher in menopausal compared to non-menopausal groups, but there were no differences in type X collagen, interleukin-6 (IL-6), and matrix metallopeptidase-13 (MMP13) between groups [Supplementary Results, Fig. S1(B)–(E)]. When we performed meta-analyses between estrogen and selective estrogen receptor modulators (SERM) treated and non-treated groups, COMP and CTX-II were lower in treated groups, while no effect was observed for IL-6 (Supplementary Results, Fig. S2).
The timing of estrogen treatment modulates beneficial effects on cartilage degeneration
Given the inconsistent outcomes of HT for treating OA
, we next tested the timing hypothesis in estrogen treatment for cartilage degeneration. When examining raw data input for the model, studies that started estrogen treatment “early” in menopause resulted in higher SMD than estrogen treatment started “late” [Fig. 4(A), Table S6]. Of the 20,000 completed simulations, 180 were deemed valid [Fig. 4(B)–(C), S3(A)]. The average values of a and b were 0.0089 [95% CI: 0.0003, 0.0162] and −0.1337 [95% CI: −0.2168, −0.0539], respectively [Fig. 4(D, E)]. Based on our simulations, both a and b were non-zero, suggesting a relationship exists between the timing of starting estrogen treatment and degree of cartilage degeneration. Specifically, treatment started “early” in menopause leads to improvement in cartilage quality while estrogen treatment started “late” in menopause may not affect cartilage. Of the included studies, only one manuscript included evaluation of multiple time points
Effects of ovariectomy and estrogen therapy on type II collagen degradation and structural integrity of articular cartilage in rats: implications of the time of initiation.
, and the relationship observed is consistent with our model.
Fig. 4Simulations reveal that estradiol (E2) treatment that is started earlier after menopause onset results in more beneficial effects to cartilage than E2 treatment that is started later after the onset of menopause. A) Raw data that were included in the simulations. Each point represents the standardized mean difference (SMD) between the E2 treated menopause group and non-E2 treated menopause group from an individual time point from each study. Input data are listed in Table S6. B) Model details and constraints are outlined. Code used for simulations is available in Appendix S3. C) The valid simulations are displayed, with the average a and b values. D) The distribution of a values generated from valid models. The shaded region indicates the 95% confidence interval (CI). E) The distribution of b values generated from valid models. The shaded region indicates the 95% CI. F) Sensitivity analysis that propagates error throughout the model.
We next examined how error propagated through the model [Fig. 4(F)]. Even with a 20% error in both a and b, the total difference in SMD was ∼1.5. This finding suggests even with large errors, our model is robust and supports the conclusion that there is a relationship between the timing of starting estrogen treatment and degree of cartilage degeneration. In a series of sensitivity analyses, we observed that results remained consistent when using a linear model and when controlling for species and rigor; however, simulations were not robust when controlling for joint under study [Supplementary Results, Figs. S3(B)–(H)].
Lastly, given the grouping of data into distinct “early” and “late” clusters with limited spread across time [Fig. 4(A)], we also evaluated this relationship using a sub-group meta-analysis. The overall effect size of estrogen treatment (regardless of timing) was −2.697 (95% CI [−3.554, −1.841]) with high heterogeneity (I2 = 82.2). Conversely, the effect size of early estrogen treatment was −3.654 (95% CI [−4.945, −2.364]) with moderate heterogeneity (I2 = 52.2), and the effect size of late estrogen treatment was −1.525 (95% CI [−2.032, −1.019]) with low heterogeneity (I2 = 28.9). This finding further supports that there may be an effect of timing of initiation of estrogen treatment on cartilage degeneration.
Estrogen dosage modulates beneficial effects on cartilage degeneration
One of the goals of pharmaceutical design is establishing the optimal dose that provides maximal therapeutic benefits while minimizing toxicity. Thus, we next interrogated the impact of estrogen dosage on cartilage degeneration. Estrogen treatment at higher doses (>500 μg/kg/day) resulted in greater improvements in cartilage degeneration than estrogen treatment at lower doses (<500 μg/kg/day) [Fig. 5(A), Table S6]. Of the 20,000 simulations, 19,999 were deemed valid [Fig. 5(B)–(C), S4(A)]. The average values of a and b were found to be non-zero, with a = −0.00000127 [95% CI: −2.658 × 10−6, −0.1459 × 10−6] and b = 0.0037 [95% CI: 0.0003, 0.0069]. The distribution of simulated values for a and b are shown in Fig. 5(D) and (E), respectively. These results suggest there is a relationship between dose of estrogen and cartilage integrity, with higher doses of estrogen likely leading to improved cartilage integrity. The same trends were observed in the two included studies that compared multiple doses of estrogen.
Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM).
Fig. 5Simulations reveal that higher doses of estradiol (E2) treatment result in more beneficial effects to cartilage than lower doses. A) Raw data that were included in the simulations. Each point represents the standardized mean difference (SMD) between the E2 treated menopause group and non-E2 treated menopause group at each dose from each study. Input data are listed in Table S6. B) Model details and constraints are outlined. Code used for simulations is available in Appendix S4. C) The valid simulations are displayed, with the average a and b values. D) The distribution of a values generated from valid models. The shaded region indicates the 95% confidence interval (CI). E) The distribution of b values generated from valid models. The shaded region indicates the 95% CI. F) Sensitivity analysis that propagates error throughout the model.
We next checked how error propagated through the model [Fig. 5(F)]. We found a 20% error in both a and b resulted in a difference in SMD of ∼6. Given the range of SMDs, relatively small errors would likely alter the conclusions generated from this model. As such, conclusions drawn from this set of simulations should be approached cautiously. Given most studies used relatively low doses of estrogen, we tested the robustness of the model by including only doses less than 500 μg/kg/day [Fig. S5(A)]. In these simulations, a and b were both non-zero, suggesting that there is a relationship between estrogen dosing and cartilage degeneration even at lower doses. In our sensitivity analyses, we observed that results remained consistent when using a linear model [Figs. S4(B)–(C)], including only studies that utilized rats, including only studies that utilized knees, and including only studies that were rigorous [Supplementary Results, Figs. S5(B)–(E)]. Lastly, when attempting to combine the dosage and timing data into a multiple linear regression model, we found data were too sparse for meaningful conclusions to be drawn (Supplementary Results, Fig. S6).
and previous preclinical work, we performed a systematic review of menopausal animal models used to study OA, meta-analyses of overlapping metrics of cartilage integrity, and a series of mathematical models to begin evaluating the efficacy of estrogen treatment for OA. Through these analyses, the following conclusions can be made: (1) menopausal aging results in worse cartilage degeneration compared to non-menopausal aging, (2) there are shortcomings in the translatability and rigor of the current preclinical studies aimed at understanding OA and menopause, (3) the menopausal effects on cartilage are more complex than diminished estrogen signaling alone, and (4) the dosage and the timing of initiation of estrogen treatment may modulate therapeutic effects on cartilage.
OVX was the most utilized method for inducing menopause. OVX is a simple model resulting in a drop in estrogen; however, there are several limitations in translatability. First, in natural menopause, ovarian tissue remains intact, and levels of other ovarian hormones, such as testosterone, remain unchanged
. In contrast, removal of the ovaries leads to loss of all ovarian hormones, including testosterone, which likely has effects on tissue homeostasis. Second, humans experience perimenopause, which is the gradual transition from regular menstrual cycling to a postmenopausal state
. However, OVX models do not recapitulate the perimenopausal state. In the clinic, young women who undergo oophorectomy and older women who undergo natural menopause are considered distinct clinical populations with separate presentations and medical needs
. Therefore, modeling natural menopause using OVX in young animals is unlikely to recapitulate the human condition and limits the usefulness of these models.
A few of the included studies utilized ER-KO animals or estrogen-receptor antagonists to model menopausal effects on OA
. However, this model does not account for all menopause-induced effects, and this is best illustrated by the inability of estrogen replacement to treat all menopause-related symptoms
. We found ER-KO animals consistently had less severe changes in cartilage compared to OVX animals. More recent work has demonstrated other menopause-associated changes, such as the marked increase in follicle stimulating hormone (FSH), can affect chondrocyte health
, we completed a rigor and reproducibility analysis. Unfortunately, we found that ARRIVE scores have not been improving over time. We recommend defining inclusion and exclusion criteria a priori and reporting animal care and monitoring protocols, as these were unsatisfactorily reported in the majority of included studies. Additionally, we recommend authors register their study protocols
and provide statements regarding availability of complete datasets. Increased attention to ensuring rigor and reproducibility is needed for accurate interpretation of presented findings and to improve the translatability of preclinical studies.
In other diseases associated with menopause onset, estrogen treatment potential is modulated by different factors, including tissue type, co-administration of progesterone, timing of treatment initiation, patient age, route of administration, type of estrogen, dosage, and duration of use
. Using an ordinary differential equation (ODE) solver, we identified a relationship between the timing of estrogen treatment initiation and cartilage integrity. Specifically, estrogen treatment started “early” (day 0 after OVX) appeared to improve cartilage integrity compared to non-treated animals, while estrogen treatment started “late” (days 20–30 after OVX) did not seem to have effects. These findings support the timing hypothesis as an explanation for the effects of estrogen treatment on OA and may explain some of the inconsistencies in the success of HT for treating OA in humans
. Given the limited number of studies, we could not control for many of the other aforementioned variables that likely affect the therapeutic potential of estrogen. Thus, we highlight the multi-factorial network of estrogen treatment on cartilage as an important topic for future studies.
Although this review adds context to the growing literature examining the effects of menopause on OA, it does have limitations. Here, we focused on outcomes related to articular cartilage, though it is understood that OA is a disease of the entire joint
, there is a need for more holistic studies that examine tissue–tissue interactions within menopause-associated OA. In examining the translational potential of this work, our findings are limited to menopause in the context of cis-gendered, female bodies, though we acknowledge HT is common among other populations including trans-gender individuals. Additionally, given the limited number of studies as well as species and joint heterogeneity, many of our analyses were likely underpowered, and the numerical coefficients generated in our models cannot be interpreted as a direct representation of the observed relationships. Instead, these models provide preliminary evidence for the presence or absence of a potential relationship between timing and dosage of estrogen and cartilage degeneration. There were also a limited number of studies that investigated treatment started late in menopause and/or using higher doses of estrogen. Therefore, caution should be used in interpreting these findings, and further investigation into these critical gaps is needed. Clinically, estrogen is rarely prescribed as a monotherapy
but rather in combination with progesterone. None of the studies identified in our review included treatment with progesterone, thus it is unclear whether the observations reported here translate to HT. Lastly, due to limited data available, we were unable to generate a robust model evaluating the effects of timing and dosage simultaneously, thereby limiting our understanding of this likely important interaction. Additional original research studies focused specifically on elucidating the relationship between timing and dosage of estrogen treatment in the context of OA are needed.
Conclusions
In this work, we summarized the current state-of-the-science aimed towards understanding how menopause affects OA in preclinical models. Most studies utilized young rodents under OVX conditions. To improve clinical relevance, we recommend the incorporation of aging and natural menopause models into the study of OA. Additionally, future studies should investigate the role of non-estrogen mediated hormonal changes that occur with menopause and are likely to play a role in the development of OA. Our mathematical models identified a potential relationship between cartilage degeneration and the timing of estrogen treatment initiation as well as estrogen dosage. To confirm the validity of these models, original research studies directly investigating these relationships and interactions are needed. Lastly, rigor and reproducibility of published literature in this area has not improved over the last 20 years. There is a clear need for scientists to be more intentional in incorporating the principles outlined in the ARRIVE Guidelines
All authors made substantial contributions in the following areas: (1) conception and design of the study, acquisition of data, analysis and interpretation of data, drafting of the article; (2) final approval of the article to be submitted; and (3) agreement to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy are appropriately investigated, resolved, and the resolution documented in the literature.
The specific contributions of the authors are as follows:
GG provided the concept, idea and experimental design for the studies. GG and FA wrote the manuscript. GG, ACB, HI, NJ, RCT, and FA provided data collection, analyses, and interpretation of the data. GG, ACB, HI, NJ, RCT, and FA reviewed, edited, and approved the final version of the manuscript. GG and FA obtained funding for the studies.
Conflict of interest statement
The authors have no financial support or other benefits from commercial sources for the work reported in the manuscript, or any other financial interests that could create a potential conflict of interest or the appearance of a conflict of interest with regard to the work.
Acknowledgments
This study was supported by (1) the Pitt Integrated Clinical and Geroscience Research Training Program (Grant Number: 5T32AG021885-19) for GG, (2) the University of Pittsburgh Clinical and Translational Science Scholars Program (KL2 TR001856) for ACB, and (3) the Interdisciplinary Mentoring and Research in Women's Cardiovascular Health (Grant Number: K24HL123565) for RCT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors would like to acknowledge the contributions, mentoring, and expertise from Dr. Christopher Evans. Lastly, the authors would like to thank Li Wang and Phillip Grosse at the University of Pittsburgh's Clinical and Translational Science Institute (CTSI) for their statistical consultation (Grant Number: UL1-TR-001857).
In comparison to age-matched, non-menopausal controls, some metrics of cartilage degeneration were significantly worse in menopausal groups. A) Meta analysis of histological score of cartilage degeneration including only studies that blinded analyses and randomized animals to each group. B) Meta analysis of type X collagen expression. C) Meta analysis of matrix metallopeptidase 13 (MMP13). D) Meta-analysis of interleukin-6 expression (IL-6). E) Meta-analysis of urinary cartilage oligomeric matrix protein (COMP).
Estradiol and selective estrogen receptor modulator (SERM) treatment affected some but not all markers of cartilage degeneration. A) Meta-analysis of interleukin-6 (IL-6) expression for estradiol treatment versus no treatment. B) Meta-analysis of urinary cartilage oligomeric matrix protein (COMP) for estradiol treatment versus no treatment. C) Meta analysis of urinary c-terminal cross-linked telopeptide of type II collagen (CTX-II) for estradiol treatment versus no treatment. D) Meta-analysis of IL-6 expression for SERM treatment versus no treatment. E) Meta-analysis of urinary COMP for SERM treatment versus no treatment.
Supplemental results from timing simulations. A) Distribution of root mean squared error (RMSE) values from quadratic model. B) Valid simulations generated from linear model. C) Distribution of RMSE values from linear model. D) How changes in a affect RMSE. E) How changes in b affect RMSE values. F) Simulations only including studies with mouse as the species. G) Simulations only including studies with knee as the joint under study. H) Simulations only including studies that were blinded.
Supplemental results from dosage simulations. A) Distribution of root mean squared error (RMSE) values from quadratic model. B) Valid simulations generated from linear model. C) Distribution of RMSE values from linear model. D) How changes in a affect RMSE. E) How changes in b affect RMSE values.
Sensitivity analyses for dosage studies. A) Valid simulations including only studies with a dosage less than 500 μg/kg/day. B) Simulations only including studies with mouse as the species. C) Simulations only including rats as the species. D) Simulations only including studies with knee as the joint under study and only studies that were blinded. E) Simulations only studies with hand as the joint under study.
Simulations using a combined model with both timing and dosage are inconclusive. A) Raw data included in simulation, with individual points indicating data from an individual study. B) Distribution of R2 values generated from valid models. C) Distribution of intercept values generated from valid models. D) Distribution of dosage slope values generated from valid models. E) Distribution of timing slope values generated from valid models.
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Impact of exercise on articular cartilage in people at risk of, or with established, knee osteoarthritis: a systematic review of randomised controlled trials.
Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice.
Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM).
Alterations to cell metabolism in connective tissues of the knee after ovariohysterectomy in a rabbit model: are there implications for the postmenopausal athlete?.
The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover.
Amelioration of collagen-induced arthritis and immune-associated bone loss through signaling via estrogen receptor alpha, and not estrogen receptor beta or G protein-coupled receptor 30.
Role of endogenous and exogenous female sex hormones in arthritis and osteoporosis development in B10.Q-ncf1∗/∗ mice with collagen-induced chronic arthritis.
Treatment with recombinant lubricin attenuates osteoarthritis by positive feedback loop between articular cartilage and subchondral bone in ovariectomized rats.
Combined effect of bilateral ovariectomy and anterior cruciate ligament transection with medial meniscectomy on the development of osteoarthritis model.
Effects of ovariectomy and estrogen therapy on type II collagen degradation and structural integrity of articular cartilage in rats: implications of the time of initiation.
Three-dimensional visualization and pathologic characteristics of cartilage and subchondral bone changes in the lumbar facet joint of an ovariectomized mouse model.
Combined detection of serum CTX-II and COMP concentrations in osteoarthritis model rabbits: an effective technique for early diagnosis and estimation of disease severity.
Brief report: Cartilage thickness change as an imaging biomarker of knee osteoarthritis progression: data from the Foundation for the National Institutes of Health Osteoarthritis Biomarkers Consortium.
Relationship between CTX-II and patient characteristics, patient-reported outcome, muscle strength, and rehabilitation in patients with a focal cartilage lesion of the knee: a prospective exploratory cohort study of 48 patients.
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