Advertisement

Uncovering the “riddle of femininity” in osteoarthritis: a systematic review and meta-analysis of menopausal animal models and mathematical modeling of estrogen treatment

  • G. Gilmer
    Correspondence
    Address correspondence and reprint requests to: G. Gilmer, Suite 308, Bridgeside Point Building II, 450 Technology Drive, Pittsburgh, PA 15219, USA.
    Affiliations
    Medical Scientist Training Program, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Cellular and Molecular Pathology Graduate Program, University of Pittsburgh, Pittsburgh, PA, USA

    Discovery Center for Musculoskeletal Recovery, Schoen Adams Research Institute at Spaulding, Rehabilitation Hospital, Boston, MA, USA

    Department of Physical Medicine & Rehabilitation, Harvard Medical School, Boston, MA, USA
    Search for articles by this author
  • A.C. Bean
    Affiliations
    Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA, USA

    McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
    Search for articles by this author
  • H. Iijima
    Affiliations
    Institute for Advanced Research, Nagoya University, Nagoya University, Nagoya, Japan
    Search for articles by this author
  • N. Jackson
    Affiliations
    Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
    Search for articles by this author
  • R.C. Thurston
    Affiliations
    Department of Psychiatry, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
    Search for articles by this author
  • F. Ambrosio
    Correspondence
    Address correspondence and reprint requests to: F. Ambrosio, Office 5304, 149 13th St #4002, Charlestown, MA 02129, USA.
    Affiliations
    Discovery Center for Musculoskeletal Recovery, Schoen Adams Research Institute at Spaulding, Rehabilitation Hospital, Boston, MA, USA

    Department of Physical Medicine & Rehabilitation, Harvard Medical School, Boston, MA, USA

    Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA, USA

    McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
    Search for articles by this author
Open AccessPublished:January 05, 2023DOI:https://doi.org/10.1016/j.joca.2022.12.009

      Summary

      Objective

      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.

      Keywords

      Abbreviations:

      OA (osteoarthritis), HT (hormone therapy), PECO (Population, Exposure, Comparison, Outcome), SMD (Standardized Mean Difference), SD (Standard Deviation), OARSI (Osteoarthritis Research Society International), ER-KO (Estrogen Receptor Knock Out), OVX (Overectomy), CTX-II (C-terminal cross-linked telopeptide of type II collagen), COMP (Cartilage oligomeric matrix protein), MMP13 (Matrix metallopeptidase 13), IL-6 (Interleukin-6), SERM (Selective estrogen receptor modulator), E2 (Estradiol), FSH (Follicle stimulating hormone), ODE (Ordinary differential equation), CI (Confidence interval)

      Introduction

      The “riddle of femininity” has puzzled the medical community for centuries
      • Hettlage-Varjas A.
      • Kurz C.
      [Difficulties in becoming a woman and staying a woman. On the problems of female identity in menopause].
      . Historically, medicine has defined male bodies as “normal”, with female physiology deviating from that of males viewed as a “fault”
      • Young K.
      • Fisher J.
      • Kirkman M.
      “Do mad people get endo or does endo make you mad?”: clinicians' discursive constructions of medicine and women with endometriosis.
      . 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
      • Tom Harkin O.J.S.
      • Mikulski Barbara A.
      • Waxman Henry A.
      Drug Safety: Most Drugs Withdrawn in Recent Years Had Greater Health Risks for 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.
      • Beery A.K.
      • Zucker I.
      Sex bias in neuroscience and biomedical research.
      ,
      • Karp N.A.
      • Reavey N.
      Sex bias in preclinical research and an exploration of how to change the status quo.
      Orthopedics and rehabilitation are no exception in failing to close the gaps in our understanding of female biology
      • Bryant J.
      • Yi P.
      • Miller L.
      • Peek K.
      • Lee D.
      Potential sex bias exists in orthopaedic basic science and translational research.
      . Post-menopausal women are nearly twice as likely as men to develop knee and hand osteoarthritis (OA)
      • Srikanth V.K.
      • Fryer J.L.
      • Zhai G.
      • Winzenberg T.M.
      • Hosmer D.
      • Jones G.
      A meta-analysis of sex differences prevalence, incidence and severity of osteoarthritis.
      • Cui A.
      • Li H.
      • Wang D.
      • Zhong J.
      • Chen Y.
      • Lu H.
      Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies.
      • Blagojevic M.
      • Jinks C.
      • Jeffery A.
      • Jordan K.P.
      Risk factors for onset of osteoarthritis of the knee in older adults: a systematic review and meta-analysis.
      . Yet, most OA animal models utilize only males
      • Iijima H.
      • Gilmer G.
      • Wang K.
      • Sivakumar S.
      • Evans C.
      • Matsui Y.
      • et al.
      Meta-analysis integrated with multi-omics data analysis to elucidate pathogenic mechanisms of age-related knee osteoarthritis in mice.
      . Surprisingly, in our recent study investigating OA in both sexes, male mice developed more severe OA compared to female mice
      • Iijima H.
      • Gilmer G.
      • Wang K.
      • Bean A.
      • He Y.
      • Lin H.
      • et al.
      Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity.
      , which is antithetical to what is observed in humans
      • Katz J.N.
      • Arant K.R.
      • Loeser R.F.
      Diagnosis and treatment of hip and knee osteoarthritis: a review.
      . 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
      • Diaz Brinton R.
      Minireview: translational animal models of human menopause: challenges and emerging opportunities.
      . 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
      • Felson D.T.
      • Neogi T.
      Emerging treatment models in rheumatology: challenges for osteoarthritis trials.
      . When considering treatments for other menopause-associated symptoms, hormone therapy (HT) remains the gold standard for treatment
      • Paciuc J.
      Hormone therapy in menopause.
      ,
      The 2017 hormone therapy position statement of the North American Menopause Society.
      . However, clinical reports of the efficacy of HT for treating OA and joint pain remain inconclusive
      The 2017 hormone therapy position statement of the North American Menopause Society.
      • Xiao Y.P.
      • Tian F.M.
      • Dai M.W.
      • Wang W.Y.
      • Shao L.T.
      • Zhang L.
      Are estrogen-related drugs new alternatives for the management of osteoarthritis?.
      • de Klerk B.M.
      • Schiphof D.
      • Groeneveld F.P.
      • Koes B.W.
      • Osch G.J.V.M. van
      • Meurs J.B.J.
      • et al.
      Limited evidence for a protective effect of unopposed oestrogen therapy for osteoarthritis of the hip: a systematic review.
      • Watt F.E.
      Hand osteoarthritis, menopause and menopausal hormone therapy.
      . One theory for the inconsistent results observed in HT is the “timing hypothesis”
      • “The Hormone Therapy Position Statement of The North American Menopause Society” Advisory P
      The 2022 hormone therapy position statement of The North American Menopause Society.
      . The timing hypothesis suggests that HT may exert differential effects according to whether it is administered in the early or late stages of menopause
      • “The Hormone Therapy Position Statement of The North American Menopause Society” Advisory P
      The 2022 hormone therapy position statement of The North American Menopause Society.
      . 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
      • Mehta J.M.
      • Chester R.C.
      • Kling J.M.
      The timing hypothesis: hormone therapy for treating symptomatic women during menopause and its relationship to cardiovascular disease.
      and dementia
      • Rocca W.A.
      • Grossardt B.R.
      • Shuster L.T.
      Oophorectomy, estrogen, and dementia: a 2014 update.
      , while if started later in menopause, pathologic effects are observed
      • Paciuc J.
      Hormone therapy in menopause.
      . Given these effects are tissue-specific
      The 2017 hormone therapy position statement of the North American Menopause Society.
      , 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
      • Sniekers Y.H.
      • Weinans H.
      • Bierma-Zeinstra S.M.
      • van Leeuwen J.P.
      • van Osch G.J.
      Animal models for osteoarthritis: the effect of ovariectomy and estrogen treatment - a systematic approach.
      . 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
      • Sniekers Y.H.
      • Weinans H.
      • Bierma-Zeinstra S.M.
      • van Leeuwen J.P.
      • van Osch G.J.
      Animal models for osteoarthritis: the effect of ovariectomy and estrogen treatment - a systematic approach.
      . 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
      The 2017 hormone therapy position statement of the North American Menopause Society.
      , 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)
      • Moher D.
      • Liberati A.
      • Tetzlaff J.
      • Altman D.G.
      • Group P.
      Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement.
      ,
      • Shamseer L.
      • Moher D.
      • Clarke M.
      • Ghersi D.
      • Liberati A.
      • Petticrewet M.
      • et al.
      Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation.
      , Meta-Analysis of Observational Studies in Epidemiology (MOOSE) checklist (Appendix S2)
      • Stroup D.F.
      • Berlin J.A.
      • Morton S.C.
      • Olkin I.
      • Williamson G.D.
      • Rennie D.
      • et al.
      Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group.
      , Cochrane Handbook for Systematic Reviews of Interventions
      • Higgins J.P.T.
      • Thomas J.
      • Chandler J.
      • Cumpston M.
      • Li T.
      • Page M.J.
      • et al.
      , and the practical guide for meta-analysis in animal studies
      • Vesterinen H.M.
      • Sena E.S.
      • Egan K.J.
      • Hirst T.C.
      • Churolov L.
      • Currie G.L.
      • et al.
      Meta-analysis of data from animal studies: a practical guide.
      . This systematic review was not registered a priori on PROSPERO
      • Page M.J.
      • Shamseer L.
      • Tricco A.C.
      Registration of systematic reviews in PROSPERO: 30,000 records and counting.
      . Eligible manuscripts were identified using a pre-defined PECO (Population, Exposure, Comparison, Outcome) criteria (Table S1)
      • Methley A.M.
      • Campbell S.
      • Chew-Graham C.
      • McNally R.
      • Cheraghi-Sohi S.
      PICO, PICOS and SPIDER: a comparison study of specificity and sensitivity in three search tools for qualitative systematic reviews.
      . Our database searches identified articles that studied the effects of menopause (defined as loss of responsiveness to ovarian hormones) on predefined articular cartilage
      • Sophia Fox A.J.
      • Bedi A.
      • Rodeo S.A.
      The basic science of articular cartilage: structure, composition, and function.
      outcomes in female animals. Predefined cartilage outcomes were generated from previous systematic reviews on articular cartilage and OA (Table S1)
      • Iijima H.
      • Gilmer G.
      • Wang K.
      • Sivakumar S.
      • Evans C.
      • Matsui Y.
      • et al.
      Meta-analysis integrated with multi-omics data analysis to elucidate pathogenic mechanisms of age-related knee osteoarthritis in mice.
      ,
      • Rongen J.J.
      • Hannink G.
      • van Tienen T.G.
      • van Luijk J.
      • Hooijmans C.R.
      The protective effect of meniscus allograft transplantation on articular cartilage: a systematic review of animal studies.
      ,
      • Bricca A.
      • Juhl C.B.
      • Steultjens M.
      • Wirth W.
      • Roos E.M.
      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
      • Higgins J.P.T.
      • Thomas J.
      • Chandler J.
      • Cumpston M.
      • Li T.
      • Page M.J.
      • et al.
      . After full-text screening, a citation search was performed on the included articles using Web of Science
      • Higgins J.P.T.
      • Thomas J.
      • Chandler J.
      • Cumpston M.
      • Li T.
      • Page M.J.
      • et al.
      . 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
      • Iijima H.
      • Gilmer G.
      • Wang K.
      • Sivakumar S.
      • Evans C.
      • Matsui Y.
      • et al.
      Meta-analysis integrated with multi-omics data analysis to elucidate pathogenic mechanisms of age-related knee osteoarthritis in mice.
      ,
      • Drevon D.
      • Fursa S.R.
      • Malcolm A.L.
      Intercoder reliability and validity of WebPlotDigitizer in extracting graphed data.
      ,
      • A. R. WebPlotDigitizer
      . 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
      • Percie du Sert N.
      • Hurst V.
      • Ahluwalia A.
      • Alam S.
      • Avey M.T.
      • Baker M.
      • et al.
      The ARRIVE guidelines 2.0: updated guidelines for reporting animal research.
      . 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
      • Higgins J.P.T.
      • Thomas J.
      • Chandler J.
      • Cumpston M.
      • Li T.
      • Page M.J.
      • et al.
      . 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)):
      SMD=ax2+bx+c
      (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)
      • da Silva J.A.
      • Colville-Nash P.
      • Spector T.D.
      • Scott D.L.
      • Willoughby D.A.
      Inflammation-induced cartilage degradation in female rodents. Protective role of sex hormones.
      • Da Silva J.A.
      • Larbre J.P.
      • Seed M.P.
      • Cutolo M.
      • Villaggio B.
      • Scott D.L.
      • et al.
      Sex differences in inflammation induced cartilage damage in rodents. The influence of sex steroids.
      • Turner A.S.
      • Athanasiou K.A.
      • Zhu C.F.
      • Alvis M.R.
      • Bryant H.U.
      Biochemical effects of estrogen on articular cartilage in ovariectomized sheep.
      • Chambers M.G.
      • Cox L.
      • Chong L.
      • Suri N.
      • Cover P.
      • Bayliss M.T.
      • et al.
      Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice.
      • Hoegh-Andersen P.
      • Tanko L.B.
      • Andersen T.L.
      • Lundberg C.V.
      • Mo J.A.
      • Heegaard A.
      • et al.
      Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application.
      • Christgau S.
      • Tanko L.B.
      • Cloos P.A.
      • Mouritzen U.
      • Christiansen C.
      • Delaissé J.
      • et al.
      Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM).
      • Jochems C.
      • Islander U.
      • Erlandsson M.
      • Verdrengh M.
      • Ohlsson C.
      • Carlsten H.
      Osteoporosis in experimental postmenopausal polyarthritis: the relative contributions of estrogen deficiency and inflammation.
      • Wang Y.
      • Liu Z.
      • Wang Q.
      • Feng Q.
      • Chen W.
      Early detection of tibial cartilage degradation and cancellous bone loss in an ovariectomized rat model.
      • Sondergaard B.C.
      • Oestergaard S.
      • Christiansen C.
      • Tanko L.B.
      • Karsdal M.A.
      The effect of oral calcitonin on cartilage turnover and surface erosion in an ovariectomized rat model.
      • Hart D.A.
      • Achari Y.
      Alterations to cell metabolism in connective tissues of the knee after ovariohysterectomy in a rabbit model: are there implications for the postmenopausal athlete?.
      • Sniekers Y.H.
      • van Osch G.J.
      • Ederveen A.G.
      • Inzunza J.
      • Gustafsson J.
      • van Leeuwen J.P.T.M.
      • et al.
      Development of osteoarthritic features in estrogen receptor knockout mice.
      • Bay-Jensen A.C.
      • Tabassi N.C.
      • Sondergaard L.V.
      • Andersen T.L.
      • Dagnaes-Hansen F.
      • Garnero P.
      • et al.
      The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover.
      • Engdahl C.
      • Jochems C.
      • Windahl S.H.
      • Börjesson A.E.
      • Ohlsson C.
      • Carlsten H.
      • et al.
      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.
      • Sniekers Y.H.
      • Weinans H.
      • van Osch G.J.
      • van Leeuwen J.P.
      Oestrogen is important for maintenance of cartilage and subchondral bone in a murine model of knee osteoarthritis.
      • Jochems C.
      • Islander U.
      • Erlandsson M.
      • Engdahl C.
      • Lagerquist M.
      • Gjertsson I.
      • et al.
      Role of endogenous and exogenous female sex hormones in arthritis and osteoporosis development in B10.Q-ncf1∗/∗ mice with collagen-induced chronic arthritis.
      • Bay-Jensen A.C.
      • Nielsen R.H.
      • Segovia-Silvestre T.
      • Azria M.
      • Staedtler F.
      • Letzkus M.
      • et al.
      A microarray analysis of full depth knee cartilage of ovariectomized rats.
      • Jochems C.
      • Islander U.
      • Erlandsson M.
      • Engdahl C.
      • Lagerquist M.
      • Ohlsson C.
      • et al.
      Effects of oestradiol and raloxifene on the induction and effector phases of experimental postmenopausal arthritis and secondary osteoporosis.
      • Islander U.
      • Jochems C.
      • Stubelius A.
      • Andersson A.
      • Lagerquist M.K.
      • Ohlsson C.
      • et al.
      Combined treatment with dexamethasone and raloxifene totally abrogates osteoporosis and joint destruction in experimental postmenopausal arthritis.
      • Engdahl C.
      • Borjesson A.E.
      • Forsman H.F.
      • Andersson A.
      • Stubelius A.
      • Krust A.
      • et al.
      The role of total and cartilage-specific estrogen receptor alpha expression for the ameliorating effect of estrogen treatment on arthritis.
      • Wang Q.
      • Liu Z.
      • Wang Y.
      • Pan Q.
      • Feng Q.
      • Huang Q.
      • et al.
      Quantitative ultrasound assessment of cartilage degeneration in ovariectomized rats with low estrogen levels.
      • Simas J.M.
      • Kunz R.I.
      • Brancalhao R.M.
      • Ribeiro Lde F.
      • Bertolini G.R.
      Effects of physical exercise on the cartilage of ovariectomized rats submitted to immobilization.
      • Cui Z.
      • Xu C.
      • Li X.
      • Song J.
      • Yu B.
      Treatment with recombinant lubricin attenuates osteoarthritis by positive feedback loop between articular cartilage and subchondral bone in ovariectomized rats.
      • Kim J.L.
      • Moon C.W.
      • Son Y.S.
      • Kim S.J.
      Combined effect of bilateral ovariectomy and anterior cruciate ligament transection with medial meniscectomy on the development of osteoarthritis model.
      • Oestergaard S.
      • Sondergaard B.C.
      • Hoegh-Andersen P.
      • Henriksen K.
      • Qvist P.
      • Christiansen C.
      • et al.
      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.
      • Li S.
      • Niu G.
      • Wu Y.
      • Du G.
      • Huang C.
      • Yin X.
      • et al.
      Vitamin D prevents articular cartilage erosion by regulating collagen II turnover through TGF-beta1 in ovariectomized rats.
      • Chen H.
      • Zhu H.
      • Zhang K.
      • Chen K.
      • Yang H.
      Estrogen deficiency accelerates lumbar facet joints arthritis.
      • Wu T.
      • Ni S.
      • Cao Y.
      • Liao S.
      • Hu J.
      • Duan C.
      Three-dimensional visualization and pathologic characteristics of cartilage and subchondral bone changes in the lumbar facet joint of an ovariectomized mouse model.
      • Zhou J.
      • Zhong P.
      • Liao Y.
      • Liu J.
      • Liao Y.
      • Xie H.
      • et al.
      Electroacupuncture ameliorates subchondral bone deterioration and inhibits cartilage degeneration in ovariectomised rats.
      • Xu X.
      • Li X.
      • Liang Y.
      • Ou Y.
      • Huang J.
      • Xiong J.
      • et al.
      Estrogen modulates cartilage and subchondral bone remodeling in an ovariectomized rat model of postmenopausal osteoarthritis.
      • Yang H.J.
      • Kim M.J.
      • Qiu J.Y.
      • Zhang T.
      • Wu X.
      • Jang D.
      • et al.
      Rice porridge containing Welsh onion root water extract alleviates osteoarthritis-related pain behaviors, glucose levels, and bone metabolism in osteoarthritis-induced ovariectomized rats.
      • Sasono B.
      • Rantam F.A.
      • Suroto H.
      • Notobroto H.B.
      • Am A.
      The effect of estrogen on type 2 collagen levels in the joint cartilage of post-menopausal murine subjects.
      • Kiyomoto K.
      • Iba K.
      • Hanaka M.
      • Ibe K.
      • Hayakawa H.
      • Teramoto A.
      • et al.
      High bone turnover state under osteoporotic changes induces pain-like behaviors in mild osteoarthritis model mice.
      • Toda T.
      • Sugioka Y.
      • Koike T.
      Soybean isoflavone can protect against osteoarthritis in ovariectomized rats.
      • Mansoori M.N.
      • Raghuvanshi A.
      • Shukla P.
      • Awasthi P.
      • Trivedi R.
      • Goel A.
      • et al.
      Medicarpin prevents arthritis in post-menopausal conditions by arresting the expansion of TH17 cells and pro-inflammatory cytokines.
      • Jin L.Y.
      • Lv Z.D.
      • Su X.J.
      • Xu S.
      • Liu H.Y.
      • Li X.F.
      Region-specific effects of blocking estrogen receptors on longitudinal bone growth.
      • Ziemian S.N.
      • Ayobami O.O.
      • Rooney A.M.
      • Kelly N.H.
      • Holyoak D.T.
      • Ross F.P.
      • et al.
      Low bone mass resulting from impaired estrogen signaling in bone increases severity of load-induced osteoarthritis in female mice.
      . 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. 1
      Fig. 1Our systematic review resulted in 36 articles 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 .
      We next assessed the rigor and reproducibility of the included studies using the ARRIVE Guidelines 2.0 [Fig. 2(A), Table S4]
      • Percie du Sert N.
      • Hurst V.
      • Ahluwalia A.
      • Alam S.
      • Avey M.T.
      • Baker M.
      • et al.
      The ARRIVE guidelines 2.0: updated guidelines for reporting animal research.
      . 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. 2
      Fig. 2The literature aimed at understanding menopause and osteoarthritis in preclinical models has not become more rigorous or reproducible 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. 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
      • Hame S.L.
      • Alexander R.A.
      Knee osteoarthritis in women.
      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. 3
      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
      • Bai B.
      • Li Y.
      Combined detection of serum CTX-II and COMP concentrations in osteoarthritis model rabbits: an effective technique for early diagnosis and estimation of disease severity.
      • Bihlet A.R.
      • Byrjalsen I.
      • Bay-Jensen A.C.
      • Andersen J.R.
      • Christiansen C.
      • Riis B.J.
      • et al.
      Associations between biomarkers of bone and cartilage turnover, gender, pain categories and radiographic severity in knee osteoarthritis.
      • Eckstein F.
      • Collins J.E.
      • Nevitt M.C.
      • Lynch J.A.
      • Kraus V.B.
      • Katz J.N.
      • et al.
      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.
      , was increased in menopausal compared to non-menopausal animals [Fig. 3(E)]. CTX-II is known to change with aging
      • Rotterud J.H.
      • Reinholt F.P.
      • Beckstrom K.J.
      • Risberg M.A.
      • Aroen A.
      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.
      . However, given all but one study included young animals (PMID: 17665432
      • Sondergaard B.C.
      • Oestergaard S.
      • Christiansen C.
      • Tanko L.B.
      • Karsdal M.A.
      The effect of oral calcitonin on cartilage turnover and surface erosion in an ovariectomized rat model.
      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
      • “The Hormone Therapy Position Statement of The North American Menopause Society” Advisory P
      The 2022 hormone therapy position statement of The North American Menopause Society.
      , 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
      • Oestergaard S.
      • Sondergaard B.C.
      • Hoegh-Andersen P.
      • Henriksen K.
      • Qvist P.
      • Christiansen C.
      • et al.
      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. 4
      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 . B) Model details and constraints are outlined. Code used for simulations is available in . 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.
      • Hoegh-Andersen P.
      • Tanko L.B.
      • Andersen T.L.
      • Lundberg C.V.
      • Mo J.A.
      • Heegaard A.
      • et al.
      Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application.
      ,
      • Christgau S.
      • Tanko L.B.
      • Cloos P.A.
      • Mouritzen U.
      • Christiansen C.
      • Delaissé J.
      • et al.
      Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM).
      Fig. 5
      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 . B) Model details and constraints are outlined. Code used for simulations is available in . 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).

      Discussion

      With the increasing incidence of OA
      • Zhang Y.
      • Jordan J.M.
      Epidemiology of osteoarthritis.
      and disproportionate effects on post-menopausal women
      • Hame S.L.
      • Alexander R.A.
      Knee osteoarthritis in women.
      , there is a growing need to understand how menopause propagates OA pathogenesis
      • Hame S.L.
      • Alexander R.A.
      Knee osteoarthritis in women.
      . Building upon the latest systematic review
      • Sniekers Y.H.
      • Weinans H.
      • Bierma-Zeinstra S.M.
      • van Leeuwen J.P.
      • van Osch G.J.
      Animal models for osteoarthritis: the effect of ovariectomy and estrogen treatment - a systematic approach.
      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
      • Diaz Brinton R.
      Minireview: translational animal models of human menopause: challenges and emerging opportunities.
      . 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
      • Santoro N.
      Perimenopause: from research to practice.
      . 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
      The 2017 hormone therapy position statement of the North American Menopause Society.
      ,
      • Hendrix S.L.
      Bilateral oophorectomy and premature menopause.
      . 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
      • Sniekers Y.H.
      • van Osch G.J.
      • Ederveen A.G.
      • Inzunza J.
      • Gustafsson J.
      • van Leeuwen J.P.T.M.
      • et al.
      Development of osteoarthritic features in estrogen receptor knockout mice.
      ,
      • Jin L.Y.
      • Lv Z.D.
      • Su X.J.
      • Xu S.
      • Liu H.Y.
      • Li X.F.
      Region-specific effects of blocking estrogen receptors on longitudinal bone growth.
      ,
      • Ziemian S.N.
      • Ayobami O.O.
      • Rooney A.M.
      • Kelly N.H.
      • Holyoak D.T.
      • Ross F.P.
      • et al.
      Low bone mass resulting from impaired estrogen signaling in bone increases severity of load-induced osteoarthritis in female mice.
      . 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
      The 2017 hormone therapy position statement of the North American Menopause Society.
      . 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
      • Huan Z.
      • Wang Y.
      • Zhang M.
      • Zhang X.
      • Liu Y.
      • Kong L.
      • et al.
      Follicle-stimulating hormone worsens osteoarthritis by causing inflammation and chondrocyte dedifferentiation.
      • Wang Y.
      • Zhang M.
      • Huan Z.
      • Shao S.
      • Zhang X.
      • Kong D.
      • et al.
      FSH directly regulates chondrocyte dedifferentiation and cartilage development.
      • Kong D.
      • Guan Q.
      • Li G.
      • Xin W.
      • Qi X.
      • Guo Y.
      • et al.
      Expression of FSHR in chondrocytes and the effect of FSH on chondrocytes.
      . These findings further support the need for studies investigating mechanisms of menopause-induced OA beyond the role of estrogen.
      Using the ARRIVE Guidelines
      • Percie du Sert N.
      • Hurst V.
      • Ahluwalia A.
      • Alam S.
      • Avey M.T.
      • Baker M.
      • et al.
      The ARRIVE guidelines 2.0: updated guidelines for reporting animal research.
      , 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
      • Bert B.
      • Heinl C.
      • Chmielewska J.
      • Schwarz F.
      • Grune B.
      • Hensel A.
      • et al.
      Refining animal research: the Animal Study Registry.
      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
      The 2017 hormone therapy position statement of the North American Menopause Society.
      . 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
      • Xiao Y.P.
      • Tian F.M.
      • Dai M.W.
      • Wang W.Y.
      • Shao L.T.
      • Zhang L.
      Are estrogen-related drugs new alternatives for the management of osteoarthritis?.
      • de Klerk B.M.
      • Schiphof D.
      • Groeneveld F.P.
      • Koes B.W.
      • Osch G.J.V.M. van
      • Meurs J.B.J.
      • et al.
      Limited evidence for a protective effect of unopposed oestrogen therapy for osteoarthritis of the hip: a systematic review.
      • Watt F.E.
      Hand osteoarthritis, menopause and menopausal hormone therapy.
      ,
      • Tanamas S.K.
      • Wijethilake P.
      • Wluka A.E.
      • Schwarz F.
      • Grune B.
      • Hensel A.
      • et al.
      Sex hormones and structural changes in osteoarthritis: a systematic review.
      . 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
      • Poole A.R.
      Osteoarthritis as a whole joint disease.
      . Given the plethora of studies showing effects of menopause on other joint tissues such as bone
      • Ji M.X.
      • Yu Q.
      Primary osteoporosis in postmenopausal women.
      , 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
      The 2017 hormone therapy position statement of the North American Menopause Society.
      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
      • Percie du Sert N.
      • Hurst V.
      • Ahluwalia A.
      • Alam S.
      • Avey M.T.
      • Baker M.
      • et al.
      The ARRIVE guidelines 2.0: updated guidelines for reporting animal research.
      in future studies.

      Author contributions

      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).

      Appendix A. Supplementary data

      The following are the Supplementary data to this article. Raw codes shown in Appendicies S3-S5 are available here: https://github.com/gggilmer/KneeOsteoarthritisMenopauseReviewCodes2023.
      • Fig. S1

        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).

      • Fig. S2

        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.

      • Fig. S3

        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.

      • Fig. S4

        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.

      • Fig. S5

        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.

      • Fig. S6

        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.

      References

        • Hettlage-Varjas A.
        • Kurz C.
        [Difficulties in becoming a woman and staying a woman. On the problems of female identity in menopause].
        Psyche (Stuttg). 1995; 49: 903-937
        • Young K.
        • Fisher J.
        • Kirkman M.
        “Do mad people get endo or does endo make you mad?”: clinicians' discursive constructions of medicine and women with endometriosis.
        Fem Psychol. 2019; 29: 337-356
        • Tom Harkin O.J.S.
        • Mikulski Barbara A.
        • Waxman Henry A.
        Drug Safety: Most Drugs Withdrawn in Recent Years Had Greater Health Risks for Women.
        2001 (Accessed)
        • Beery A.K.
        • Zucker I.
        Sex bias in neuroscience and biomedical research.
        Neurosci Biobehav Rev. 2011; 35: 565-572
        • Karp N.A.
        • Reavey N.
        Sex bias in preclinical research and an exploration of how to change the status quo.
        Br J Pharmacol. 2019; 176: 4107-4118
        • Bryant J.
        • Yi P.
        • Miller L.
        • Peek K.
        • Lee D.
        Potential sex bias exists in orthopaedic basic science and translational research.
        J Bone Joint Surg Am. 2018; 100: 124-130
        • Srikanth V.K.
        • Fryer J.L.
        • Zhai G.
        • Winzenberg T.M.
        • Hosmer D.
        • Jones G.
        A meta-analysis of sex differences prevalence, incidence and severity of osteoarthritis.
        Osteoarthritis Cartilage. 2005; 13: 769-781
        • Cui A.
        • Li H.
        • Wang D.
        • Zhong J.
        • Chen Y.
        • Lu H.
        Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies.
        EClinicalMedicine. 2020; 29–30100587
        • Blagojevic M.
        • Jinks C.
        • Jeffery A.
        • Jordan K.P.
        Risk factors for onset of osteoarthritis of the knee in older adults: a systematic review and meta-analysis.
        Osteoarthritis Cartilage. 2010; 18: 24-33
        • Iijima H.
        • Gilmer G.
        • Wang K.
        • Sivakumar S.
        • Evans C.
        • Matsui Y.
        • et al.
        Meta-analysis integrated with multi-omics data analysis to elucidate pathogenic mechanisms of age-related knee osteoarthritis in mice.
        J Gerontol A Biol Sci Med Sci. 2022; 77: 1321-1334
        • Iijima H.
        • Gilmer G.
        • Wang K.
        • Bean A.
        • He Y.
        • Lin H.
        • et al.
        Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity.
        Nat Commun. 2023; 14: 18
        • Katz J.N.
        • Arant K.R.
        • Loeser R.F.
        Diagnosis and treatment of hip and knee osteoarthritis: a review.
        JAMA. 2021; 325: 568-578
        • Diaz Brinton R.
        Minireview: translational animal models of human menopause: challenges and emerging opportunities.
        Endocrinology. 2012; 153: 3571-3578
        • Felson D.T.
        • Neogi T.
        Emerging treatment models in rheumatology: challenges for osteoarthritis trials.
        Arthritis Rheumatol. 2018; 70: 1175-1181
        • Paciuc J.
        Hormone therapy in menopause.
        Adv Exp Med Biol. 2020; 1242: 89-120
      1. The 2017 hormone therapy position statement of the North American Menopause Society.
        Menopause. 2018; 25: 1362-1387
        • Xiao Y.P.
        • Tian F.M.
        • Dai M.W.
        • Wang W.Y.
        • Shao L.T.
        • Zhang L.
        Are estrogen-related drugs new alternatives for the management of osteoarthritis?.
        Arthritis Res Ther. 2016; 18: 151
        • de Klerk B.M.
        • Schiphof D.
        • Groeneveld F.P.
        • Koes B.W.
        • Osch G.J.V.M. van
        • Meurs J.B.J.
        • et al.
        Limited evidence for a protective effect of unopposed oestrogen therapy for osteoarthritis of the hip: a systematic review.
        Rheumatology. 2009; 48: 104-112
        • Watt F.E.
        Hand osteoarthritis, menopause and menopausal hormone therapy.
        Maturitas. 2016; 83: 13-18
        • “The Hormone Therapy Position Statement of The North American Menopause Society” Advisory P
        The 2022 hormone therapy position statement of The North American Menopause Society.
        Menopause. 2022; 29: 767-794
        • Mehta J.M.
        • Chester R.C.
        • Kling J.M.
        The timing hypothesis: hormone therapy for treating symptomatic women during menopause and its relationship to cardiovascular disease.
        J Womens Health (Larchmt). 2019; 28: 705-711
        • Rocca W.A.
        • Grossardt B.R.
        • Shuster L.T.
        Oophorectomy, estrogen, and dementia: a 2014 update.
        Mol Cell Endocrinol. 2014; 389: 7-12
        • Sniekers Y.H.
        • Weinans H.
        • Bierma-Zeinstra S.M.
        • van Leeuwen J.P.
        • van Osch G.J.
        Animal models for osteoarthritis: the effect of ovariectomy and estrogen treatment - a systematic approach.
        Osteoarthritis Cartilage. 2008; 16: 533-541
        • Moher D.
        • Liberati A.
        • Tetzlaff J.
        • Altman D.G.
        • Group P.
        Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement.
        Ann Intern Med. 2009; 151 (W264): 264-269
        • Shamseer L.
        • Moher D.
        • Clarke M.
        • Ghersi D.
        • Liberati A.
        • Petticrewet M.
        • et al.
        Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation.
        BMJ. 2015; 350: g7647
        • Stroup D.F.
        • Berlin J.A.
        • Morton S.C.
        • Olkin I.
        • Williamson G.D.
        • Rennie D.
        • et al.
        Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group.
        JAMA. 2000; 283: 2008-2012
        • Higgins J.P.T.
        • Thomas J.
        • Chandler J.
        • Cumpston M.
        • Li T.
        • Page M.J.
        • et al.
        Cochrane Handbook for Systematic Reviews of Interventions. 2022 (6.3 ed)
        • Vesterinen H.M.
        • Sena E.S.
        • Egan K.J.
        • Hirst T.C.
        • Churolov L.
        • Currie G.L.
        • et al.
        Meta-analysis of data from animal studies: a practical guide.
        J Neurosci Methods. 2014; 221: 92-102
        • Page M.J.
        • Shamseer L.
        • Tricco A.C.
        Registration of systematic reviews in PROSPERO: 30,000 records and counting.
        Syst Rev. 2018; 7: 32
        • Methley A.M.
        • Campbell S.
        • Chew-Graham C.
        • McNally R.
        • Cheraghi-Sohi S.
        PICO, PICOS and SPIDER: a comparison study of specificity and sensitivity in three search tools for qualitative systematic reviews.
        BMC Health Serv Res. 2014; 14: 579
        • Sophia Fox A.J.
        • Bedi A.
        • Rodeo S.A.
        The basic science of articular cartilage: structure, composition, and function.
        Sports Health. 2009; 1: 461-468
        • Rongen J.J.
        • Hannink G.
        • van Tienen T.G.
        • van Luijk J.
        • Hooijmans C.R.
        The protective effect of meniscus allograft transplantation on articular cartilage: a systematic review of animal studies.
        Osteoarthritis Cartilage. 2015; 23: 1242-1253
        • Bricca A.
        • Juhl C.B.
        • Steultjens M.
        • Wirth W.
        • Roos E.M.
        Impact of exercise on articular cartilage in people at risk of, or with established, knee osteoarthritis: a systematic review of randomised controlled trials.
        Br J Sports Med. 2019; 53: 940-947
        • Drevon D.
        • Fursa S.R.
        • Malcolm A.L.
        Intercoder reliability and validity of WebPlotDigitizer in extracting graphed data.
        Behav Modif. 2017; 41: 323-339
        • A. R. WebPlotDigitizer
        (Accessed)
        • Percie du Sert N.
        • Hurst V.
        • Ahluwalia A.
        • Alam S.
        • Avey M.T.
        • Baker M.
        • et al.
        The ARRIVE guidelines 2.0: updated guidelines for reporting animal research.
        Br J Pharmacol. 2020; 177: 3617-3624
        • da Silva J.A.
        • Colville-Nash P.
        • Spector T.D.
        • Scott D.L.
        • Willoughby D.A.
        Inflammation-induced cartilage degradation in female rodents. Protective role of sex hormones.
        Arthritis Rheum. 1993; 36: 1007-1013
        • Da Silva J.A.
        • Larbre J.P.
        • Seed M.P.
        • Cutolo M.
        • Villaggio B.
        • Scott D.L.
        • et al.
        Sex differences in inflammation induced cartilage damage in rodents. The influence of sex steroids.
        J Rheumatol. 1994; 21: 330-337
        • Turner A.S.
        • Athanasiou K.A.
        • Zhu C.F.
        • Alvis M.R.
        • Bryant H.U.
        Biochemical effects of estrogen on articular cartilage in ovariectomized sheep.
        Osteoarthritis Cartilage. 1997; 5: 63-69
        • Chambers M.G.
        • Cox L.
        • Chong L.
        • Suri N.
        • Cover P.
        • Bayliss M.T.
        • et al.
        Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice.
        Arthritis Rheum. 2001; 44: 1455-1465
        • Hoegh-Andersen P.
        • Tanko L.B.
        • Andersen T.L.
        • Lundberg C.V.
        • Mo J.A.
        • Heegaard A.
        • et al.
        Ovariectomized rats as a model of postmenopausal osteoarthritis: validation and application.
        Arthritis Res Ther. 2004; 6: R169-R180
        • Christgau S.
        • Tanko L.B.
        • Cloos P.A.
        • Mouritzen U.
        • Christiansen C.
        • Delaissé J.
        • et al.
        Suppression of elevated cartilage turnover in postmenopausal women and in ovariectomized rats by estrogen and a selective estrogen-receptor modulator (SERM).
        Menopause. 2004; 11: 508-518
        • Jochems C.
        • Islander U.
        • Erlandsson M.
        • Verdrengh M.
        • Ohlsson C.
        • Carlsten H.
        Osteoporosis in experimental postmenopausal polyarthritis: the relative contributions of estrogen deficiency and inflammation.
        Arthritis Res Ther. 2005; 7: R837-R843
        • Wang Y.
        • Liu Z.
        • Wang Q.
        • Feng Q.
        • Chen W.
        Early detection of tibial cartilage degradation and cancellous bone loss in an ovariectomized rat model.
        Biomed Res Int. 2017; 20179654056
        • Sondergaard B.C.
        • Oestergaard S.
        • Christiansen C.
        • Tanko L.B.
        • Karsdal M.A.
        The effect of oral calcitonin on cartilage turnover and surface erosion in an ovariectomized rat model.
        Arthritis Rheum. 2007; 56: 2674-2678
        • Hart D.A.
        • Achari Y.
        Alterations to cell metabolism in connective tissues of the knee after ovariohysterectomy in a rabbit model: are there implications for the postmenopausal athlete?.
        Br J Sports Med. 2010; 44: 867-871
        • Sniekers Y.H.
        • van Osch G.J.
        • Ederveen A.G.
        • Inzunza J.
        • Gustafsson J.
        • van Leeuwen J.P.T.M.
        • et al.
        Development of osteoarthritic features in estrogen receptor knockout mice.
        Osteoarthritis Cartilage. 2009; 17: 1356-1361
        • Bay-Jensen A.C.
        • Tabassi N.C.
        • Sondergaard L.V.
        • Andersen T.L.
        • Dagnaes-Hansen F.
        • Garnero P.
        • et al.
        The response to oestrogen deprivation of the cartilage collagen degradation marker, CTX-II, is unique compared with other markers of collagen turnover.
        Arthritis Res Ther. 2009; 11: R9
        • Engdahl C.
        • Jochems C.
        • Windahl S.H.
        • Börjesson A.E.
        • Ohlsson C.
        • Carlsten H.
        • et al.
        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.
        Arthritis Rheum. 2010; 62: 524-533
        • Sniekers Y.H.
        • Weinans H.
        • van Osch G.J.
        • van Leeuwen J.P.
        Oestrogen is important for maintenance of cartilage and subchondral bone in a murine model of knee osteoarthritis.
        Arthritis Res Ther. 2010; 12: R182
        • Jochems C.
        • Islander U.
        • Erlandsson M.
        • Engdahl C.
        • Lagerquist M.
        • Gjertsson I.
        • et al.
        Role of endogenous and exogenous female sex hormones in arthritis and osteoporosis development in B10.Q-ncf1∗/∗ mice with collagen-induced chronic arthritis.
        BMC Musculoskelet Disord. 2010; 11: 284
        • Bay-Jensen A.C.
        • Nielsen R.H.
        • Segovia-Silvestre T.
        • Azria M.
        • Staedtler F.
        • Letzkus M.
        • et al.
        A microarray analysis of full depth knee cartilage of ovariectomized rats.
        BMC Res Notes. 2011; 4: 63
        • Jochems C.
        • Islander U.
        • Erlandsson M.
        • Engdahl C.
        • Lagerquist M.
        • Ohlsson C.
        • et al.
        Effects of oestradiol and raloxifene on the induction and effector phases of experimental postmenopausal arthritis and secondary osteoporosis.
        Clin Exp Immunol. 2011; 165: 121-129
        • Islander U.
        • Jochems C.
        • Stubelius A.
        • Andersson A.
        • Lagerquist M.K.
        • Ohlsson C.
        • et al.
        Combined treatment with dexamethasone and raloxifene totally abrogates osteoporosis and joint destruction in experimental postmenopausal arthritis.
        Arthritis Res Ther. 2011; 13: R96
        • Engdahl C.
        • Borjesson A.E.
        • Forsman H.F.
        • Andersson A.
        • Stubelius A.
        • Krust A.
        • et al.
        The role of total and cartilage-specific estrogen receptor alpha expression for the ameliorating effect of estrogen treatment on arthritis.
        Arthritis Res Ther. 2014; 16: R150
        • Wang Q.
        • Liu Z.
        • Wang Y.
        • Pan Q.
        • Feng Q.
        • Huang Q.
        • et al.
        Quantitative ultrasound assessment of cartilage degeneration in ovariectomized rats with low estrogen levels.
        Ultrasound Med Biol. 2016; 42: 290-298
        • Simas J.M.
        • Kunz R.I.
        • Brancalhao R.M.
        • Ribeiro Lde F.
        • Bertolini G.R.
        Effects of physical exercise on the cartilage of ovariectomized rats submitted to immobilization.
        Einstein (Sao Paulo). 2015; 13: 574-579
        • Cui Z.
        • Xu C.
        • Li X.
        • Song J.
        • Yu B.
        Treatment with recombinant lubricin attenuates osteoarthritis by positive feedback loop between articular cartilage and subchondral bone in ovariectomized rats.
        Bone. 2015; 74: 37-47
        • Kim J.L.
        • Moon C.W.
        • Son Y.S.
        • Kim S.J.
        Combined effect of bilateral ovariectomy and anterior cruciate ligament transection with medial meniscectomy on the development of osteoarthritis model.
        Ann Rehabil Med. 2016; 40: 583-591
        • Oestergaard S.
        • Sondergaard B.C.
        • Hoegh-Andersen P.
        • Henriksen K.
        • Qvist P.
        • Christiansen C.
        • et al.
        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.
        Arthritis Rheum. 2006; 54: 2441-2451
        • Li S.
        • Niu G.
        • Wu Y.
        • Du G.
        • Huang C.
        • Yin X.
        • et al.
        Vitamin D prevents articular cartilage erosion by regulating collagen II turnover through TGF-beta1 in ovariectomized rats.
        Osteoarthritis Cartilage. 2016; 24: 345-353
        • Chen H.
        • Zhu H.
        • Zhang K.
        • Chen K.
        • Yang H.
        Estrogen deficiency accelerates lumbar facet joints arthritis.
        Sci Rep. 2017; 7: 1379
        • Wu T.
        • Ni S.
        • Cao Y.
        • Liao S.
        • Hu J.
        • Duan C.
        Three-dimensional visualization and pathologic characteristics of cartilage and subchondral bone changes in the lumbar facet joint of an ovariectomized mouse model.
        Spine J. 2018; 18: 663-673
        • Zhou J.
        • Zhong P.
        • Liao Y.
        • Liu J.
        • Liao Y.
        • Xie H.
        • et al.
        Electroacupuncture ameliorates subchondral bone deterioration and inhibits cartilage degeneration in ovariectomised rats.
        Acupunct Med. 2018; 36: 37-43
        • Xu X.
        • Li X.
        • Liang Y.
        • Ou Y.
        • Huang J.
        • Xiong J.
        • et al.
        Estrogen modulates cartilage and subchondral bone remodeling in an ovariectomized rat model of postmenopausal osteoarthritis.
        Med Sci Monit. 2019; 25: 3146-3153
        • Yang H.J.
        • Kim M.J.
        • Qiu J.Y.
        • Zhang T.
        • Wu X.
        • Jang D.
        • et al.
        Rice porridge containing Welsh onion root water extract alleviates osteoarthritis-related pain behaviors, glucose levels, and bone metabolism in osteoarthritis-induced ovariectomized rats.
        Nutrients. 2019; 11: 2019
        • Sasono B.
        • Rantam F.A.
        • Suroto H.
        • Notobroto H.B.
        • Am A.
        The effect of estrogen on type 2 collagen levels in the joint cartilage of post-menopausal murine subjects.
        J Hard Tissue Biol. 2019; 28: 245-249
        • Kiyomoto K.
        • Iba K.
        • Hanaka M.
        • Ibe K.
        • Hayakawa H.
        • Teramoto A.
        • et al.
        High bone turnover state under osteoporotic changes induces pain-like behaviors in mild osteoarthritis model mice.
        J Bone Miner Metab. 2020; 38: 806-818
        • Toda T.
        • Sugioka Y.
        • Koike T.
        Soybean isoflavone can protect against osteoarthritis in ovariectomized rats.
        J Food Sci Technol. 2020; 57: 3409-3414
        • Mansoori M.N.
        • Raghuvanshi A.
        • Shukla P.
        • Awasthi P.
        • Trivedi R.
        • Goel A.
        • et al.
        Medicarpin prevents arthritis in post-menopausal conditions by arresting the expansion of TH17 cells and pro-inflammatory cytokines.
        Int Immunopharmacol. 2020; 82: 106299
        • Jin L.Y.
        • Lv Z.D.
        • Su X.J.
        • Xu S.
        • Liu H.Y.
        • Li X.F.
        Region-specific effects of blocking estrogen receptors on longitudinal bone growth.
        J Endocrinol. 2021; 250: 13-24
        • Ziemian S.N.
        • Ayobami O.O.
        • Rooney A.M.
        • Kelly N.H.
        • Holyoak D.T.
        • Ross F.P.
        • et al.
        Low bone mass resulting from impaired estrogen signaling in bone increases severity of load-induced osteoarthritis in female mice.
        Bone. 2021; 152116071
        • NIH
        Rigor and Reproducibility.
        2022 (Accessed)
        • Hame S.L.
        • Alexander R.A.
        Knee osteoarthritis in women.
        Curr Rev Musculoskelet Med. 2013; 6: 182-187
        • Bai B.
        • Li Y.
        Combined detection of serum CTX-II and COMP concentrations in osteoarthritis model rabbits: an effective technique for early diagnosis and estimation of disease severity.
        J Orthop Surg Res. 2016; 11: 149
        • Bihlet A.R.
        • Byrjalsen I.
        • Bay-Jensen A.C.
        • Andersen J.R.
        • Christiansen C.
        • Riis B.J.
        • et al.
        Associations between biomarkers of bone and cartilage turnover, gender, pain categories and radiographic severity in knee osteoarthritis.
        Arthritis Res Ther. 2019; 21: 203
        • Eckstein F.
        • Collins J.E.
        • Nevitt M.C.
        • Lynch J.A.
        • Kraus V.B.
        • Katz J.N.
        • et al.
        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.
        Arthritis Rheumatol. 2015; 67: 3184-3189
        • Rotterud J.H.
        • Reinholt F.P.
        • Beckstrom K.J.
        • Risberg M.A.
        • Aroen A.
        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.
        BMC Musculoskelet Disord. 2014; 15: 99
        • Zhang Y.
        • Jordan J.M.
        Epidemiology of osteoarthritis.
        Clin Geriatr Med. 2010; 26: 355-369
        • Santoro N.
        Perimenopause: from research to practice.
        J Womens Health (Larchmt). 2016; 25: 332-339
        • Hendrix S.L.
        Bilateral oophorectomy and premature menopause.
        Am J Med. 2005; 118: 131-135
        • Huan Z.
        • Wang Y.
        • Zhang M.
        • Zhang X.
        • Liu Y.
        • Kong L.
        • et al.
        Follicle-stimulating hormone worsens osteoarthritis by causing inflammation and chondrocyte dedifferentiation.
        FEBS Open Bio. 2021; 11: 2292-2303
        • Wang Y.
        • Zhang M.
        • Huan Z.
        • Shao S.
        • Zhang X.
        • Kong D.
        • et al.
        FSH directly regulates chondrocyte dedifferentiation and cartilage development.
        J Endocrinol. 2021; 248: 193-206
        • Kong D.
        • Guan Q.
        • Li G.
        • Xin W.
        • Qi X.
        • Guo Y.
        • et al.
        Expression of FSHR in chondrocytes and the effect of FSH on chondrocytes.
        Biochem Biophys Res Commun. 2018; 495: 587-593
        • Bert B.
        • Heinl C.
        • Chmielewska J.
        • Schwarz F.
        • Grune B.
        • Hensel A.
        • et al.
        Refining animal research: the Animal Study Registry.
        PLoS Biol. 2019; 17e3000463
        • Tanamas S.K.
        • Wijethilake P.
        • Wluka A.E.
        • Schwarz F.
        • Grune B.
        • Hensel A.
        • et al.
        Sex hormones and structural changes in osteoarthritis: a systematic review.
        Maturitas. 2011; 69: 141-156
        • Poole A.R.
        Osteoarthritis as a whole joint disease.
        HSS J. 2012; 8: 4-6
        • Ji M.X.
        • Yu Q.
        Primary osteoporosis in postmenopausal women.
        Chronic Dis Transl Med. 2015; 1: 9-13