This systematic review summarizes evidence for associations between female reproductive factors  and intraocular pressure (IOP) or open-angle glaucoma (OAG).Understanding the associations between female reproductive factors and glaucoma may shed light on the disease pathogenesis and aid clinical prediction and personalized treatment strategies. Importantly, some factors are modifiable, which may lead to new therapies.Two reviewers independently extracted articles in MEDLINE, Embase, Cochrane Database of Systematic Reviews, and Cochrane Central Register of Controlled Trials databases to identify relevant studies. Eligibility criteria included studies with human subjects aged > 18 years; a measured outcome of either IOP or OAG; a cohort, case-control, cross-sectional, or randomized controlled trial design; a reported measure of association, such as the hazard ratio, relative risk, odds ratio, or mean difference, with an associated confidence interval; and a measured exposure of at least 1 of the following variables: age at menarche, parity, OC use, age at menopause, or PMH use.We included a total of 27 studies. Substantial differences in study designs, exposure and treatment levels, treatment durations, and variable reporting precluded a meaningful quantitative synthesis of the identified studies. Overall, relatively consistent associations between PMH use and a lower IOP were identified. Estrogen-only PMH use may be associated with lower OAG risk, which may be modified by race. No significant associations were found with combined estrogen-and-progesterone PMH use. No strong associations between parity or age at menarche and glaucoma were found, but a younger age at menopause was associated with an increased glaucoma risk, and adverse associations were identified with a longer duration of OC use, though no overall association with OC use was found.The association between PMH use and lower IOP or OAG risk is a potentially clinically relevant and modifiable risk factor and should be investigated further, although this needs to be interpreted in the context of a high risk of bias across included studies. Future research should examine associations with IOP specifically and how the relationship between genetic factors and OAG risks may be influenced by female reproductive factors. Clinical studies have also found that exogenous estrogen supplementation (in the form of hormone therapy) for individuals in hypoestrogenic states such as menopause may be associated with a lower risk of POAG18 and lower IOP values,30 although the specific mechanism is unclear.A number of epidemiologic studies provide further support for the potential associations between estrogen-associated female reproductive factors and glaucoma.This systematic review summarizes the associations between female reproductive factors  with IOP and open-angle glaucoma . Clarifying the associations between female reproductive factors and glaucoma (and related traits) may provide a better understanding of the sex-specific risk factors for OAG and may serve as a foundation for future preventative and therapeutic strategies in its management.31 No predefined protocol for this systematic review has been published previously. As this study involved only a review and synthesis of existing literature, it was exempt from institutional review board approval. All research adhered to the principles outlined in the Declaration of Helsinki.We conducted a systematic review to assess and summarize the associations between key female reproductive factors  and IOP or OAG in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines.www.ophthalmologyglaucoma.org). We also performed a search of the Google Scholar database, hand-searched the reference lists of all included studies and relevant review articles, and contacted information specialists and clinical experts to identify potential additional studies not captured by our original database searches.We conducted a systematic search of the MEDLINE, Embase, Cochrane Database of Systematic Reviews, and Cochrane Central Register of Controlled Trials databases from inception to September 15, 2020, using keywords and medical subject heading terms relating to prespecified female reproductive factors and IOP and OAG. The search was conducted without restrictions on the time period, publication type, or language of publication. The detailed search strategies for MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials are provided in www.ophthalmologyglaucoma.org); (4) the study employs a cohort, case-control, cross-sectional, before-and-after analysis, or randomized controlled trial (RCT) design; and (5) the study reports measures of association, including the hazard ratio (HR), relative risk, odds ratio (OR), or mean difference, with associated confidence intervals (CIs) or standard errors, or sufficient data are provided to allow for calculation of these measures. We excluded studies that (1) were review articles, animal studies, case reports, conference presentations, conference abstracts, letters to the editor, or basic science studies ; (2) did not specifically report either IOP or OAG data as an outcome; (3) did not perform an ophthalmic examination or use findings from a previous examination to confirm the OAG diagnosis; or (4) were published in a non-English language.For inclusion into the systematic review, a study needed to meet the following criteria: (1) study participants were aged >18 years; (2) the study measured and reports at least 1 of the following female reproductive factors: age at menarche, parity, gravidity, OC use, age at menopause, duration of reproductive years, or PMH use; (3) the study reports either or both of the outcomes of IOP (measured in mmHg) or OAG, confirmed by an ophthalmic assessment, review of a previous assessment reporting structural or functional findings consistent with OAG , or record linkage to a previously documented diagnosis of OAG . Each reviewer independently extracted study results, which were exported as .RIS or .XLSX files into independent Excel spreadsheet forms, which were then consolidated. Discrepancies between reviewers were resolved by discussion between the 2 reviewers to achieve consensus and by consultation with a third reviewer (A.P.K.) if necessary.Title and abstract screening was performed independently by 2 reviewers (K.M.M. and S.Y.L.C.) to identify studies relevant to the associations between female reproductive factors and IOP or OAG. All titles and abstracts were imported into an Excel (Microsoft Corporation) spreadsheet to facilitate review. Studies unrelated to the exposures or outcomes of interest (as well as duplicate studies) were identified by hand and excluded at this stage. The full text of the remaining studies was then retrieved to assess the study methods and data, and studies not fulfilling all the inclusion criteria at this point were excluded  if needed.The following data were extracted from included studies: author names, year of publication, country, ethnicity of the study population, study design, number of subjects analyzed, number of eyes analyzed, determination or measurement of the specific female reproductive factor of interest and its levels, reference or control group, mean ages (for each group), specific outcome assessed , case definition of OAG, the most fully adjusted effect estimate for the association , and its corresponding CI. We also collected the adjusted covariables for each fully adjusted effect estimate. Data were extracted only from the available published articles: authors of included studies were not contacted for missing or additional information. A formal risk-of-bias assessment was conducted at both the domain level and the individual study level, modeled on the Cochrane Collaboration’s Risk-of-Bias toolThe extracted data from all included studies were arranged both by exposure  and subgroup where possible . We then assessed whether the data were suitable for quantitative summarization . Although no formal statistical tests of heterogeneity were calculated, substantial differences were found across study designs, exposure levels, treatment doses, and treatment durations, and it was noted that different studies measuring a given exposure frequently used different exposure levels as a reference. A consensus between authors determined these factors precluded a meaningful meta-analysis.41A total of 4516 articles were initially identified. After removal of duplicates, 3730 articles remained, which underwent title and abstract screening by 2 authors (K.M.M. and S.Y.L.C.). The full texts of 89 articles were retrieved for review. Of these articles, 5 were non-English; 8 reported outcomes irrelevant to this review; 17 examined irrelevant exposures; 33 were either review articles, animal studies, or basic science studies; and 1 was a duplicate study, and these were excluded. A careful examination of the reference lists of the included articles identified 2 additional relevant studies that met the inclusion criteria and were ultimately included in this review for a total of 27 studies. Characteristics of the included studies are described in This review includes studies with different designs and therefore required the use of > 1 risk-of-bias tool. Sources of bias varied according to the study design, outcome, and risk-of-bias tool used and, as such, were not directly comparable. Results of the risk-of-bias assessment are presented separately based on the study design and outcome of interest . The forFor controlled studies with IOP as the outcome, the overall risk of bias was moderate, and it was serious for uncontrolled studies on IOP. This was particularly influenced by a serious risk of bias in the confounding domain. When studies on IOP with a moderate or serious risk of bias were excluded, the remaining studies with a low risk of bias  identified that participants using an estrogen-only PMH had significantly lower IOP values compared with placebo controls and a lower risk of high-tension POAG . The overall risk of bias was moderate for associations with OAG and high for associations with IOP.24 Four of these studies were cross-sectional in design24 and included a total of 14 294 individuals with 560 OAG cases, and 1 study15 was a prospective cohort study describing 808 incident POAG cases over 1 288 200 person-years of follow-up  was associated with twice the risk of POAG  compared with achieving menarche at an age < 13 years in 1 study,34 that included 487 cases from 2573 subjects, and 1 was a cohort study of 333 POAG cases observed over 619 356 person-years of follow-up15 , with prolonged use of OC (> 5 years) associated with a 25% higher risk . Other studies evaluating the relationship between any use of or ever using OC and OAG did not identify any overall association.34Four included studies explored the association between OC use and OAG risks. Three were cross-sectional studieslow-up15 . Data fr35 Four of these35 were cross-sectional  and 1 study15 was a prospective cohort study with a total of 806 incident POAG cases discovered during 1 280 918 person-years of follow-up  in a cross-sectional population study in Korea.35 A significant trend between an increasing number of pregnancies and an increased risk of OAG was identified in another study24 (P for trend = 0.03), but no association was reported in the other 2 included studies.23 The association was inconsistent across the included studies, and results from the most highly powered study suggested no direct association between parity and OAG risks.15Five included studies reported the association between the number of pregnancies (parity) and OAG risks.ollow-up . Differe24 Five of these studies were cross-sectional in design ,24 and 2 were cohort studies16 with a total 462 cases over a combined 687 020 person-years of follow-up 19 and 3.5 .21The cohort studies found that women with earlier menopause had a 1.6-times higher risk of all-cause glaucoma ,21 When the analysis was limited only to women with a reported age at natural menopause, earlier menopause was associated with more than twice the risk of OAG in 3 of the included studies.22 When stratified on chronologic age, older postmenopausal women (over age of 65) who underwent menopause later than age 54 had nearly half the risk of POAG of similar-aged women with earlier menopause in a secondary analysis of the NHS cohort.14 No significant association between OAG with a history of elevated IOP (> 21 mmHg) and age at menopause was identified in the 2 studies examining that specific relationship22 , 1 study found that women undergoing earlier menopause (before 53 years of age) had a significantly higher risk of OAG than those who underwent menopause after the age of 53 .onship22 . Overall24 , but no association was identified in other cross-sectional studies or in the NHS, which was the largest and most highly powered study of this association.14Four studies  provided specific data on the total number of years of reproductive duration  and risks of POAG24 . Each ad17 2 studies18 were cohort studies  and, as such, are discussed separately from the cross-sectional studies that examined the use of unspecified preparations of PMHs as the main exposure of interest.18 No overall association was found between estrogen-only PMH use and OAG risks in the Women’s Health Initiative Sight Exam Study trial , although a prespecified analysis found the risk of incident OAG to be lower in Black women using an estrogen-only PMH compared with placebo , and this association was not identified in White participants.17Among the RCT and cohort studies examining different formulations of PMHs, estrogen-only PMH use was examined in a total 1412 individuals in 3 studies . Estroge18 although a subgroup analysis of the NHS data found that current use of a combined PMH was associated with a slightly lower risk of high-tension POAG (defined as IOP > 21 mmHg).14 There was no significant association between PMH use and OAG in the 4 cross-sectional studies.No overall association between combined estrogen-and-progesterone PMH use and OAG risks was identified in any of these 3 studies,41 Five studies were controlled studies37  compared with controls in both studies. Four studies evaluated a combined estrogen-plus-progesterone PMH user group against a control group.37 A total of 1584 eyes of individuals receiving a combined estrogen-plus-progesterone PMH were compared against a total 1469 control eyes. Two studies identified lower IOP values in the combined PMH group than in the control group27 . The largest placebo-controlled RCT to date on the association between PMH use and IOP8 found the IOP values of eyes treated with the estrogen-only PMH to be lower by 0.5 mmHg  than eyes receiving placebo after 5 months of treatment, but no difference in IOP was found in participants using combined estrogen plus progesterone over the same period of time.8 Although this study reported the difference in IOP values between treated and placebo groups for both types of PMHs independently, it did not compare the relative effects of estrogen-only to estrogen-plus-progesterone treatment.Controlled studies were further divided into those that reported analyses from estrogen-only PMH user groups and combined estrogen-plus-progesterone user groups . A total41 identified different IOP values between PMH users and nonusers, with 1 study identifying a 1.3-mmHg 40 lower IOP value in PMH users and 1 identifying a 0.4-mmHg41  higher IOP value . None of these studies compared IOP between the groups in question. The single study evaluating estrogen-only PMH use reported a 2.0-mmHg lower IOP value within participants after PMH use.26 The remaining studies examining combined PMH use found participants to have overall lower IOP after PMH use, with differences ranging from −0.7 mmHg28 to −3.8 mmHg4 lower after a course of PMH therapy.Four studies using an uncontrolled pre/post design were identified .4,26,28,Evidence from the 27 included studies highlights potentially important associations of various female reproductive factors with glaucoma and IOP. Although associations between PMHs and IOP or POAG were consistent across these studies, modest associations with other factors, including the duration of OC use and age at menopause, were reported in several studies.24 however, suggests no clear association between the age at menarche and risks of POAG. This may be owing to the inability to meta-analyze the various studies, leading to this review being underpowered to identify a true association. Although no studies directly examined the association between age at menarche and IOP, a secondary analysis of the NHS found that a later age of menarche was associated with a slightly higher risk of the normal-tension subtype of POAG (IOP < 22 mmHg),14 suggesting that a potential association between menarche age and glaucoma may occur via non–IOP-mediated mechanisms. The relationship between age at menarche and POAG should be further investigated, more completely accounting for the entire female reproductive and postreproductive history.A younger age at menarche should theoretically confer greater overall lifetime estrogen exposure, which would lead to a hypothetically lower risk of POAG. Evidence from the included observational studies,35 suggests there is likely no direct relationship between the number of term pregnancies and the risk of POAG. Although the Blue Mountains Eye Study24 and the Korean National Health and Nutrition Examination Survey (NHANES)35 reported higher numbers of pregnancies to be associated with higher risks of POAG, these results were not reproduced in other studies, and no consistent trend was found between increasing parity and increasing risks of POAG. Interestingly, no included study directly compared the risks of POAG between nulliparous women and women with any non-0 number of children, but 1 excluded study reported larger vertical neuroretinal rim widths in women of any parity than in nulliparous women, suggesting potential associations between parity and other glaucoma phenotypes.43Evidence from observational studies15 This association has been further investigated in a recent, large, US-based study , which similarly found a greater duration of OC use to be associated with a substantive increase in the odds of self-reported glaucoma and ocular hypertension compared with individuals with no history of OC use. As this study used self-reported glaucoma or ocular hypertension as outcomes, it was excluded from our systematic review, although it highlights the growing need for research around the potential associations between the duration of OC use and glaucoma and related traits. Interestingly, no associations have been identified with other glaucoma-related traits, such as visual field defects or an increased vertical cup-to-disc ratio, and OC use in secondary analyses of Wang et al20 and another excluded study.43Although the existing literature does not support a direct association between OC use and glaucoma-related traits, potential adverse associations with the duration of use have been identified. The nature of the association between ever or current OC use and OAG remains incompletely studied, but results from the NHS provide support for a potential relationship between a longer duration of use and higher POAG risks in particular.45 which may potentially explain or modify any potential associations between OC use and IOP or OAG. The inconsistent associations found among studies examining parity and OC use may be related to the age range during which OC use typically occurs versus the typically older age of glaucoma onset. This makes it challenging to identify strong and consistent associations across studies. Furthermore, no large-scale studies have been conducted examining OC use and IOP, and this remains a highly fertile area for further investigation.Decisions made by women around contraception use are influenced by numerous demographic, socioeconomic, and geographic factors,14 Although no association between the age at menopause and OAG with elevated IOP  was identified, no study directly assessed the relationship with IOP, and this represents an avenue for future investigation. Such a study may, however, prove logistically challenging, as it would require measuring IOP values before and after the menopausal transition and adjusting for age.The epidemiologic literature does not consistently support an overall association between age at menopause and POAG; however, several subgroup analyses suggest a higher risk of POAG in those with an earlier age at natural menopause. A lower risk of POAG was also found in a large subgroup analysis of older women (> 65 years) who underwent menopause at a later age, suggesting that a longer duration of estrogen exposure may reduce the POAG risk.46 but the specific effects of each are not yet fully understood.47 The number of studies reporting each of these subtypes individually did not make a subanalysis realistic in this review, although an effort was made to do so. A recent genetic risk score of 18 single nucleotide polymorphisms strongly associated with the age at natural menopause  was not found to be associated with POAG,48 suggesting no likely underlying genetic association for the findings from the included observational studies. Interestingly, 1 included study found that individuals who underwent surgically induced menopause  before the age of 43 had 1.6 times the risk of glaucoma (broadly defined) compared with a referent group of age-matched women who did not undergo oophorectomy . The association with OAG specifically was not significant, although this particular analysis was likely underpowered.16 This study examined the youngest population of menopausal women (under age 43) and was the only study to specifically assess surgically induced  menopause. Although confounding by indication for surgery cannot be entirely ruled out, this study’s finding that menopause induced at an extremely early age may increase the risk of glaucoma lends further support to the role estrogen deficiency may play in glaucoma pathophysiology.Menopause can occur naturally or can be induced by surgery or radiation. Each of these types of menopause can influence the age at menopause,22 identified a modestly lower risk of POAG from either a later age at menopause or an earlier age at menarche. The association between the duration of reproductive years and POAG risks remains unclear, and future assessments of the role of reproductive years in glaucoma pathogenesis may benefit from the development of genetic risk scores that account more accurately for endogenous estrogen exposure.Ages at menarche and menopause are surrogate variables for years of reproductive duration, a more direct  measure of endogenous estrogen exposure. Only 1 studyEstrogen levels gradually decline throughout the reproductive years and following menopause. Postmenopausal women are, therefore, at a theoretically increased risk of glaucoma and related traits, and this risk would hypothetically be attenuated by the use of exogenous estrogen supplementation.18 whereas another cohort study found a decreased risk only for the high-tension  subtype of POAG and another study reported no overall association but found race to be a significant modifier of the association.17 In this large study , analyses stratified by race identified a significant risk reduction in OAG during the follow-up period among Black women using an estrogen-only PMH, but no association was identified in White women, suggesting race may modify the association between estrogen-only PMH use and OAG.17This review found that PMH use may be associated with lower IOP, but its impact on mitigating OAG risks remains unclear. One study found a significant association between PMH use and an overall decreased risk of POAG,41 and this association with lower IOP was more consistent among estrogen-only PMH users than among combined estrogen-plus-progesterone users.26 Any potential relationship between PMH use and lower risks of POAG might, therefore, be hypothesized to occur through an IOP-mediated pathway, potentially driven through estrogen-receptor signaling.In each of these studies, the association was only significant for users of an estrogen-only PMH. Multiple studies examined the association between PMHs and IOP, with most identifying an association between the use of PMHs and lower mean IOP values,Although this review attempted a subgroup analysis of studies based on estrogen-only or estrogen-plus-progesterone use, the doses and durations of use differed between studies, precluding a meaningful direct comparison. Furthermore, several of the included studies assessing IOP did not perform comparisons against a formal control group and, as such, provide different effect estimates on the associations (or effects of the associations) with IOP than studies with a control group. Ultimately, observations in uncontrolled samples do not allow for the estimation of relative effects that would be necessary for decision-making.Although there is variability in the quality of the included studies, the overall preponderance of evidence suggests a potential association between PMH use and lower IOP, and this warrants further investigation. Notably, evidence from RCTs and prospective studies suggests that PMH use may be associated with lower IOP, and this is further supported by similar results from other studies with less robust study designs. Although causality cannot be assessed, the association is consistent across included studies (and is consistent across studies with a low risk of bias), which strengthens the level of certainty for this potential association. These associations should be further explored in different ethnic groups, employing appropriate age- and race-matched reference groups receiving standardized doses of PMHs with similar follow-up periods. Future genetic epidemiologic studies may offer insight into race as a potential modifier of the association between estrogen-only PMH use and POAG and explore whether these relationships also apply to the association with IOP.Several important sources of heterogeneity and potential bias were identified across the included studies, which made a direct comparison of the findings challenging and ultimately precluded a formal statistical synthesis of the data. Different study designs were employed , participants were included from different countries and ethnicities, and analyses did not always adjust for the same covariables.Although most studies examining associations with OAG adjusted for age and often other socioeconomic or medical covariables, residual confounding from unmeasured covariables remains a possibility such that the identified associations may be driven by unknown or unmeasured factors. Several studies adjusted for a relatively comprehensive array of covariables, whereas others adjusted for fewer covariables  and 2. SExposure data were collected in different ways: most studies used self-reported data for female reproductive factors, whereas others used health records. Both of these approaches pose potential challenges for data integrity, as self-reported exposures can be associated with recall bias  and results from record use are directly tied to the accuracy of data entry in the first instance. This potential for bias in studies of PMH or OC use was offset in some studies by the use of prospective follow-ups confirming medication use, by linkage to pharmacy records of filled medication prescriptions, or by determining serum confirmation of estrogen levels. Exposure departures remain a distinct possibility, particularly in cross-sectional studies, but are less likely to be a substantial source of bias in the included cohort or before-and-after interventional studies because participants were either directly administered the treatment in question or had verified records confirming prescriptions were filled.50 that reportedly differ based on geography51 and may be modified by lifestyle factors and race.53 Furthermore, use of OCs and PMHs is influenced by various socioeconomic and demographic factors.55 Although no included studies assessed or reported interactions between various female reproductive factors of interest, at least 1 study adjusted for all the measured female reproductive factors in multivariable analyses.15 Potential interactions between female reproductive factors themselves and socioeconomic status (SES) represent an area for further investigation with regard to any potential associations with glaucoma or related traits.Importantly, there is also a degree of intrinsic variability within the exposures of interest themselves: women reach female reproductive health milestones at different ages57 and this is reflected in different case definition criteria employed in the included studies (Appendix SA2). Although most included studies reported POAG as the main outcome and either excluded or adjusted for secondary OAGs (including pseudoexfoliation glaucoma), several did not specifically differentiate between POAG and other OAGs, and others included subjects with probable (but not necessarily definite) POAG (see Appendix SA2). Furthermore, most studies employed a direct physical examination to diagnose POAG, whereas other studies relied on previously conducted assessments. This variability in outcome definitions may have led to unintended biases, weakening our ability to identify potential associations. The studies examining IOP were more consistently robust in outcome ascertainment, as IOP was directly measured in each study. Intraocular pressure may be influenced by corneal biomechanical properties, and these were not directly measured or accounted for in any of the included studies. Nearly all studies used Goldmann Applanation Tonometry, although 2 studies used other methods to measure IOP  and are particularly consistent in studies with the lowest risks of bias. When interpreted collectively, these findings lend additional support to the hypothesis that estrogen-associated factors are associated with glaucoma and related traits.To our knowledge, no existing systematic review has consolidated all the studies examining the associations between a comprehensive set of female reproductive factors and both IOP and the OAG risk. This review addresses the existing gap in the literature and highlights areas for future research on this wide-ranging topic. The minimization of exclusion criteria during the search strategy helped ensure the review was highly comprehensive in its inclusion of relevant studies. Furthermore, the broad search criteria employed in the search strategy contributed to the comprehensiveness of this review in capturing multiple relevant exposures and outcomes.59 estrogen-receptor signaling was a key modulator of several of these IOP-associated genes. These genetic associations further underscore the importance of better understanding the associations of female reproductive milestones with glaucoma and IOP.Studies of female reproductive attributes in relation to OAG can be particularly useful, as controlling for age can help isolate the role female reproductive aging may play in the glaucoma disease processes. Female reproductive milestones that practically all women experience can be categorized by altered estrogen levels; by these proxy measures, one can examine how altered estrogen signaling may impact glaucoma more broadly. More directly, studying female reproductive milestones in relation to glaucoma or glaucoma endophenotypes can further inform our understanding of the pathophysiology of disease. In a recent study using a bioinformatics pipeline to prioritize the many newly discovered IOP-associated genes,The observational studies included in this review are susceptible to various biases, which prevent assessments of causal associations as discussed above. Epidemiologic assessments of female reproductive factors and their potential associations are complicated by a number of factors. Menarche and menopause, for example, are exposures that occur over short time frames in different relation to the typical onset of menopause, which poses a number of challenges for data collection and analysis. For the age at menarche, data collection is particularly challenging, as this female reproductive milestone is reached many decades before glaucoma typically occurs, whereas the age at menopause is typically reached 1 to 2 decades before the onset of glaucoma. These exposures are typically ascertained by recall and, if not accurately determined, may lead to misleading results for the relations between these exposures and the risk of glaucoma. Because these exposures have a small range of values and, probably, a modest effect on the disease process, very large sample sizes are necessary for studies to be appropriately powered to detect definitive associations with glaucoma.62 it is often challenging to distinguish the effects of female reproductive aging from those of chronologic aging on glaucoma and related traits. Furthermore, certain female reproductive factors (such as age at menopause63 and parity64) may be associated with socioeconomic factors, and economic deprivation may also be associated with higher rates of glaucoma or more advanced disease at initial presentation.66 Although several studies adjusted for SES, not every study assessed this in the same manner; thus, residual confounding by SES may potentially explain some of the above-identified observational associations.As the risk for glaucoma increases with age,17Another major limitation of the included studies is that analyses were performed in predominantly White populations, limiting generalizability of these findings to other ethnic groups. This is particularly notable given that race has been identified as a possible modifier of the identified associations between PMHs and POAG.67 we hypothesize that factors that increase IOP in the general population will, in turn, increase the risk of OAG. This has been demonstrated to be the case in genetic associations: genetic determinants of IOP in general populations are also risk factors for OAG in independent case-control studies.68One limitation of many of the included studies examining POAG as the main outcome of interest is that profiling of the IOP values of the population at risk for glaucoma was not performed, which makes extrapolation of results to populations at risk somewhat more challenging. Given that the risk of developing OAG in a healthy population has been shown to increase by 16% per 1-mmHg increase in IOP,Studies that examined reproductive factors but did not mention them in the title or abstract may have been missed, but we believe our search criteria to be reasonably robust. Several important studies were excluded from this paper for using self-reported glaucoma as the outcome of interest, despite providing evidence for potentially important associations of interest to this review. Similarly, several studies only provided data for OAG overall, which prevented the analysis of whether the identified associations are specific to POAG or may be associated with other phenotypes of OAG more generally. Another weakness of this review is its exclusion of non-English articles, which may give rise to a degree of publication bias, the extent of which was not formally assessed.17). There is a clear need for additional studies on the associations between female reproductive factors and glaucoma and related traits. As the prevalence of OAG is expected to increase in the coming years, investigations into sex-specific risk factors and gene-environment interactions will be important in improving our understanding of the associations and pathogenesis of OAG, potentially leading to novel preventative measures and therapies.The findings of this systematic review provide support for potential associations between certain female reproductive factors and IOP and OAG, although the reported associations need to be interpreted in the context of high risks of bias across included studies. A longer duration of OC use may be associated with a higher risk of OAG on the basis of included studies, but it is not yet clear whether use of OCs in general increases the risk of OAG or whether this potential association is mediated by IOP. An earlier age at natural menopause may be associated with a higher OAG risk. Most notably, PMH use may be associated with a lower IOP, and the estrogen-only type of PMH may be associated with a lower risk of OAG (a relationship which may be modified by raceTable S2Table S1Appendix SA1


Protocadherin 10 plays a critical role in the central nervous system including in cell adhesion, formation and maintenance of neural circuits and synapses, regulation of actin assembly, cognitive function and tumor suppression. Additionally, Pcdh10 can serve as a non-invasive diagnostic and prognostic indicator for various cancers.Protocadherin 10 (PCDH 10), a member of the superfamily of protocadherins, is a Ca2This paper collects and reviews relevant literature in Pubmed.This review describes the latest research understanding the role of Pcdh10 in neurological disease and human cancer, highlighting the importance of scrutinizing its properties for the development of targeted therapies and identifying a need for further research to explore Pcdh10 functions in other pathways, cell types and human pathologies. Cell–cell adhesion is a basic process in the morphogenesis of multicellular organisms. Originally described as cell adhesion molecules, cadherins play a crucial role in cell recognition, cell communication, morphogenesis, cytoskeletal organization, cell migration, and neural circuit formation  is a highly genetically heterogeneous neurodevelopmental disorder characterized by impaired social communications. ASD commonly co-presents with other neurological conditions, such as epilepsy, intellectual disability or bipolar disorders. Several studies have identified Pcdh10 deletion in mice display mild impairment in their social recognition and communication responses, suggesting that Pcdh10 mutations may cause ASD-related symptoms , obsessive–compulsive disorder (OCD), major depression (MD) and schizophrenia  is one of the most common types of malignant tumors. Pcdh10 is well-known as a tumor suppressor in colorectal carcinogenesis, invasion and metastasis  is the third most common fatal form of cancer around the globe and the detailed mechanism underlying gastric carcinogenesis remains unclear. Pcdh10 expression is silenced or down-regulated in gastric cancer cells and tissues  is one of the most lethal diseases worldwide (Kamisawa et al. Previous study identified that Pcdh10 expression is silenced by methylation in pancreatic cancer cell lines, and re-expression of Pcdh10 prevents the malignant biological process of PC cells (Qiu et al. In terms of anti-tumor effects, Pcdh10 overexpression can prevent the proliferation, migration, invasion ability of pancreatic cancer cells and trigger apoptosis by activating the AKT pathway (Qiu et al. The deletion of Pcdh10 has been reported in various human tumors. In addition to the aforementioned CRC, GC, PC, cervical and endometrial cancers, Pcdh10 loss has been observed in non-small-cell lung cancer (NSCLC; (Tang et al. In support of a role for Pcdh10 as a tumor suppressor, restoration of Pcdh10 in hepatocellular carcinoma cell lines inhibits proliferation and induces cell apoptosis via suppressing PI3K/Akt signaling pathway (Ye et al. It should be noted that Pcdh10 might act as a tumor oncogene in gliomas, as it is essential for the proliferation and tumorigenicity of human glioblastoma cell lines GB2 and GB16 (Echizen et al. It is interesting to note that the methylation status of Pcdh10 may be able to predict the response of lymphomas to doxorubicin (Narayan et al. An increasing number of studies have demonstrated that Pcdh10 plays an important role in cancer. Therefore, exploring therapeutic strategies targeting Pcdh10 may be of great importance in the management of several types of tumors.Therapeutic strategies that target Pcdh10 may be relevant to CRC. Zhou et al. demonstrated that hsa_circ_0001666 functions as a tumor suppressor by directly binding miR‐576‐5p and lessening its inhibitory effect on the target gene Pcdh10, thereby inhibiting cell proliferation, metastasis, EMT progression and stemness as well as triggering apoptosis of CRC cells (Zhou et al. As previously mentioned, HOTAIR lncRNA acts as the upstream regulator of Pcdh10 in cervical hela cells (Salmeron-Barcenas et al. CircPcdh10 promotes tumor progression of pancreatic cancer by increasing hTERT expression through interacting with miR-338-3p (Zhang et al. The functions of Pcdh10, its regulatory targets and the role it plays in human pathologies, remain largely unexplored. Pcdh10 is considered to play important roles in brain development and is implicated in human neurological disorders like autism, obsessive–compulsive disorder, major depression and schizophrenia. Pcdh10 has also been implicated in a range of human cancers, acting as a tumor suppressor and playing key roles in regulating tumor growth, invasion and metastasis. In contrast, Pcdh10 has also been shown to be an oncogene for the tumorigenesis of glioblastoma. Further research is required to fully elucidate the role of Pcdh10 in neurological conditions and different types of the cancers, and determine whether Pcdh10 is implicated in any other human condition. In addition, aberrant methylations of Pcdh10 have been recognized as a non-invasive biomarker for tumor diagnosis and prognosis. Though the involvement of Pcdh10 in the pathogenesis of neural diseases and human cancers has been recently established, our understanding of the molecular functions and related signaling pathways involved is limited. Moreover, the genes targeting Pcdh10 function and the relevant molecular mechanisms involved also remain to be investigated but could provide further insights into therapeutic strategies that could be developed for the treatment of Pcdh10-regulated conditions. Our review focuses on the known conditions where Pcdh10 is disrupted and its potential as a cancer biomarker, however given the various pathways regulated by Pcdh10, further research is likely to determine its role in other conditions and further explore its potential as a non-invasive biomarker of disease.


Cancer stem cells residing in tumor mass exhibit two main properties: self-renewal and differentiation. Self-renewal is the process by which a stem cell divides to preserve its stemness traits in at least one daughter cell per division. A growing number of studies show that CSCs accomplish self-renewal by both symmetric and asymmetric cell division. Symmetric cell division produces two cancer stem cells with self-renewal abilities, while asymmetric cell division produces 2 cells with different fates (an undifferentiated cancer stem cell and a cell committed to differentiate). Differentiation is a process by which CSCs give rise to more differentiated cells incapable of sustaining tumor growth. Differentiation endows CSCs with the capacity to provide a continuous supply of heterogeneous tumor cells to maintain uninterrupted tumor growth .The identification and characterization of cancer stem cells have been a key step in cancer research. Substantial efforts are dedicated to unraveling the intricate biology of this cell fraction. CSCs possess a distinct set of functional and biological attributes that distinguish them from other non-stem cells within tumors. These cells are identified by the presence or absence of various stem cell markers that are heterogeneously distributed across different tumors. These markers include cell surface proteins , cytoplasmic enzymes , and nuclear transcription factors  .Ritter et al. show that stem cells possess the innate capacity to internalize a fluorescent double-stranded DNA through electrostatic interactions facilitated by the attraction between the negatively charged dsDNA and the positively charged stem cells. In addition, the authors indicate that certain surface proteins present in the CSCs play a pivotal role in facilitating the binding of the fluorescent DNA. These molecules possess a heparin-binding domain characterized by a cluster of Lysine/Arginine residues, thereby forming a positively charged groove. Furthermore, the internalization of the dsDNA was shown to occur through a mechanism involving caveolae and/or clathrin-dependent endocytosis and micropinocytosis. This manuscript provides evidence for the existence of a well-developed glycocalyx on CSCs, featuring a diverse array of membrane-anchored proteins associated with lipid rafts and clathrin complexes with the ability to bind DNA .CSCs of solid tumors exhibit a plethora of distinctive features including the ability to survive and form spheres under low adherent conditions, the potential to initiate tumors in immunodeficient animal models, even when inoculated in minimal quantities, and the capacity to efflux drugs owing to the overexpression of ABC drug transporters. These characteristics have been extensively studied in many types of tumors; however, little has been explored about the inner membrane composition of CSCs and the implication of these features on the functionality and behavior of CSCs. A study conducted by Bustamante-Marin and Capel, it was shown a relationship between the genetics of male stem germ cells and their response to the microenvironment. Heterozygosity of the potent modifier of tumor incidence Ter, a point mutation in the dead-end homolog 1 gene (Dnd1Ter/+) renders male germ cells susceptible to tumorigenic transformation. In 129/SvJ Dnd1Ter/+ mice, ∼80% of the unilateral teratomas arise in the left testis. Interestingly, as previously shown, the vascular architecture differences between the right and left adult testes are differentially oxygenated, and the left testes express higher levels of HIF-1α (Bustamante-Marin and Capel). Here, the authors showed that systemic exposure to hypoxia during a specific window of embryonic development of male stem germ cells 129/SvJ Dnd1Ter/+ (E13.8–E14.3) eliminated the left bias of teratoma development and increased their susceptibility to develop bilateral teratoma from 3% to 64%, evaluated as E-Cadherin clusters in both gonads. The phenotype correlated with high expression of pluripotency genes Oct4, Sox2, and Nanog, elevated activity of the Nodal signaling pathway, and suppression of germ cell mitotic arrest (Bustamante-Marin and Capel). The understanding of the influence of the testicular microenvironment on the fate of male germ cells could lead to the identification of therapeutic targets to develop successful preventions and treatments for testicular cancer.Similarly, another crucial attribute that influences the fate of cancer stem cells is the tumor microenvironment (TMA). In an interesting study by Guerrero-Rodriguez et al. reviewed the potential value of the cell surface protein CD36 as a potential target for the development of novel strategies aimed at eradicating cancer stem cells and slowing tumor growth .CSCs are clinically relevant since accumulating evidence has shown that these cell subpopulations provoke tumor progression, treatment resistance, metastasis, and recurrence. In light of the resistance that has emerged in patients towards all anti-neoplastic therapies, it becomes necessary to ascertain the “Achilles heel” of CSCs for targeting and eliminating them. These approaches include chemotherapeutic drugs, TAA-specific antibodies, and gene and cell therapies. In accordance, β signaling pathways. It is important to note that the regulation of BCSCs is complex, and the interplay of multiple signaling pathways can vary among different breast cancer subtypes . Combination therapies targeting multiple pathways and personalized medicine approaches are being explored to address BCSCs and improve breast cancer treatment outcomes.The regulation of the self-renewal of CSCs is orchestrated by specific signaling pathways. In this review article, the authors summarize the current knowledge on the main signal pathway governing breast cancer stem cell (BCSCs) self-renewal and the challenges associated with harnessing these signal pathways as therapeutics for the treatment of breast cancer. The authors discuss the key signaling pathways involved including the Wnt, NF-κB, Notch, Hedgehog, Hippo, and the TGF- Losada-Garcia et al. conducted a study to demonstrate that SFPR1 regulates canonical Wnt signaling and favors a stem cell phenotype in prostate cancer. Their findings indicate that exogenous SFRP1 treatment expands the prostate cancer stem cell fraction and elevates the expression of cancer stem cell markers and genes related to the Wnt/β-catenin pathway. The authors demonstrate that SFRP1 regulates the subpopulation of CSCs by modulating the Wnt-beta catenin signaling pathway . This study stimulates further investigations aimed at discerning the potential of SFRP1 as a target for the elimination of prostate cancer stem cells.The canonical Wnt cell signaling is regulated by a plethora of molecules including SFPR1 (secreted frizzled-related protein 1).


Cereals are a group of grasses cultivated by humans for their grain. It is from these cereal grains that the majority of all calories consumed by humans are derived. The production of these grains is the result of the development of a series of hierarchical reproductive structures that form the distinct shoot architecture of the grasses. Being spatiotemporally complex, the coordination of grass shoot development is tightly controlled by a network of genes and signals, including the key phytohormone auxin. Hormonal manipulation has therefore been identified as a promising potential approach to increasing cereal crop yields and therefore ultimately global food security. Recent work translating the substantial body of auxin research from model plants into cereal crop species is revealing the contribution of auxin biosynthesis, transport, and signalling to the development of grass shoot architecture. This review discusses this still-maturing knowledge base and examines the possibility that changes in auxin biology could have been a causative agent in the evolution of differences in shoot architecture between key grass species, or could underpin the future selective breeding of cereal crops. We review recent advances in understanding auxin biology in the grass family, and explore the possibility that changes in auxin biology led to differences in shoot architecture between grass species. Poaceae) of flowering plants includes key species grown, cultivated, and bred by humans for their grain—the cereal crops. The high yields produced by these cereals have underpinned the development and maintenance of all agrarian societies since the Neolithic revolution, and continue to be vital to global food security. Between them, the ‘trinity’ of wheat, rice, and maize are predicted to account for more than half of all human calorie consumption, with the remaining cereal crops, such as barley, millets, sorghum, rye, and oats, also contributing significantly to modern global diets  and as far back as 130 MYA ] has well-established, key roles in shoot meristem development , and AtTAR2, and the second containing a group of alliinase-related TAA genes, including AtTAR3 and AtTAR4 which have been less studied (YUC phylogeny has four main branches. Two of these contain Arabidopsis shoot-expressed YUC genes and homologues (AtYUC1/AtYUC4 in one clade and AtYUC2/AtYUC6 in the other)  , catalysed by TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family enzymes . IPyA th species , as do t species . PhylogeTAA and YUC genes identified in grasses allow us to conclude that, as in Arabidopsis, different TAA and YUC genes have specific roles within a given species. For instance, ZmYUC1, OsYUC9, and OsYUC11 have important functions in controlling auxin concentration during embryo development , but have been found to be highly expressed in the shoot  encodes a YUC protein with an essential role in inflorescence development , tassel length, spikelet pair meristems on the ear , ear length, and plant height, as well as producing spikelet meristems at atypical locations on developing male and female inflorescences and at a greatly reduced number (defective endosperm18 (de18) to produce smaller grain than the wild type, with a 40% reduction in endosperm dry mass , which produces shrunken pollen grains but no other developmental defects, suggesting that auxin synthesis is essential for proper pollen grain development  has also been identified through genetic screens. Much like the spi1 mutants, vt2 single mutant maize also exhibited significant defects in inflorescence development, producing fewer ears that were shorter and had fewer spikelets  mutant exhibits increased tillering, but decreased panicle and grain size and number, which was related to a decrease in endogenous auxin levels. TSG1 was identified as an allele of the tryptophan aminotransferase gene FISH BONE (FIB)  in the model grass species Brachypodium distachyon (Brachypodium) shows that these three genes have distinct expression domains in spike meristems that collectively resemble PIN1 expression in Arabidopsis shoot meristems, with SoPIN1 expressed in the epidermis, PIN1b in developing vascular strands, and PIN1a more broadly in the internal tissues , Whilst the phenotypic effects of single ospin1a and ospin1b knockouts were minor, double mutants have drastically reduced plant height and increased tillering  and four in the B genome (TaPIN1-6b1–TaPIN1-6b4). Meanwhile TaPIN1-7 has a single homeologue in each genome  mutants have previously been shown to exhibit a similar developmental phenotype to pin mutants, namely a defect in floral meristem initiation resulting in a pin-like inflorescence , , whereinZmAFB4/5B2 and OsAFB4/5B) and between paralogues in the same genome (e.g. OsAFB2/3A show a much higher expression level in mature inflorescences than the other rice TIR1/AFB genes). In terms of functional analyses, mutation of rice TIR1/AFB genes showed that single mutant lines of Ostir1 and Osafb2 produced shorter plants, more tillers, and fewer grains per panicle  also exhibit reduced height, increased branching, and reduced seed number  and ZmIAA20 (Barren Inflorescence4) strongly affect shoot architecture, producing tassels with a reduced number of branches and spikelets, and ears with reduced length and kernel number   and one clade C ARF gene (OsARF18) in rice also show high shoot expression. All of these lack functional investigation and appear to be prime targets for further investigation of the role of auxin signalling in shoot architecture in grasses. Only two maize genes show particularly high shoot expression, ZmARF7 and ZmARF35 . ba1 mutants exhibit reduced tillering, the same phenotype that results from the disruption of shoot-expressed rice ARF genes OsARF6 and OsARF17. This link suggests that ZmARF35 mutation would result in a similar effect and should be prioritized for functional investigation.Compared with Aux/IAAs, there is much more evidence for ARFs as regulators of shoot architecture. In rice, expression profiling of OsARF1, where antisense expression resulted in decreased shoot height  mutant line  was first identified as a central coordinator of tillering in maize, and orthologous proteins have been discovered with equivalent function in wheat (TaTB1) (HTD1 expression is induced by auxin and is a widely conserved regulator of shoot architecture (BARREN STALK (BA) genes. BA1 (Another relevant example is HIGH-TILLERING DWARF1 (HTD1) in rice. HTD1 is an essential enzyme in the synthesis of strigolactone, a molecule which has its own signalling capability and influence on shoot architecture  genes involved in auxin signalling influence the development of shoot architecture in grasses, and that differences in sequence, location of expression, and timing of expression exist between these genes. Taken together, these further build the case for such changes potentially underlying changes in shoot architecture between members of the So how does the most important hormone make the most important plants? In this review, we have attempted to ask whether innovations in auxin biology are associated with the morphological innovations in the shoot architecture of the grasses. There is currently no clear answer to this question, but there are tantalizing hints that it may be the case. The dramatic changes in PIN protein structure and complement between the grasses and their near relatives in the Poales implies a strong selective drive for novel functionalities, given that these altered structures are then highly conserved within the grasses. The expression of these genes, and the functional evidence where available, certainly implicates them in the development of shoot architecture, but it is too early to firmly associate specific changes in PIN proteins with specific morphological effects. The existence of apparent grass-specific clades of auxin biosynthesis and signalling components, and the changes in expression and function relative to known paralogues in Arabidopsis, does indicate significant auxin innovation between the grasses relative to other plant species. There are also clear examples, such as the functional differences between the orthologues VT2/FB/TaTAR2.1, that support the idea that changes in expression level or timing of auxin synthesis genes might be associated with differences in shoot architecture within the grasses.However, as we have also outlined, there remains a lack of detailed understanding of auxin biology in the ‘big three’ cereals , and little species-specific knowledge exists for other, still significant cereals, such as barley, sorghum, and millets. Future work would be best focused on the functional analysis of pathways that identify and connect changes in gene sequence, associated changes in auxin distribution and response, and causally connected changes in shoot architecture. Many studies have been discussed in this review that have produced a plethora of expression data, but such knowledge remains of limited use without associated investigation into the phenotypic effects of such differences, and the underlying presence and activity of auxin. Additionally, the systematic generation and study of single and multiple mutants for auxin-related families in key grasses, based on current knowledge from Arabidopsis, would be a rational next step. The rapid advances in the ability to perform multigene CRISPR in grass species, allowing functional redundancy to be overcome even in species with complex genomes, should make such an approach more feasible.Overall, understanding the basis of the morphological innovations in the grasses, whether these are driven by changes in auxin biology, or changes in other developmental pathways, would be hugely beneficial in determining the possibility and mechanisms for the continued improvement of grass shoot architecture, with implications for improved food security and feeding the increasing global population with reduced arable land.


Transcription factor A, mitochondrial (TFAM) is a key player in mitochondrial DNA (mtDNA) transcription and replication. It was first isolated in 1988 as an activator of mitochondrial transcription [No dedicated DNA primase has been identified in mitochondria to date. Therefore, mitochondrial transcription is believed to be responsible for the synthesis of primers for mtDNA replication. This concept mechanistically links mtDNA transcription and replication. Indeed, cells with the inactivation of key components of the mtDNA transcription apparatus  remain viable but show a loss of mtDNA . In the In many studies, similarly sized nucleoid-like structures containing DNA but little to no TFAM, or vice versa, have been observed within the same cell –19. AccoThe availability of crystal structures of TFAM in complex with LSP, mitochondrial heavy strand promoter 1 (HSP1), nonspecific DNA, and transcription initiation complexes (ICs) at LSP and HSP1 –23 have What is the molecular basis underlying the differential sensitivity of mitochondrial promoters to TFAM alterations, such as changes in TFAM abundance or its primary amino acid sequence? Early biochemical and structural studies have provided a coherent picture that attributed the higher sensitivity of LSP to TFAM C-terminal truncation and DNA bending defects to this promoter’s dependence on DNA bending for the proper alignment of the TFAM’s C-terminus with the transcription start site ,21,23. HAnswers to these and other pertinent questions should greatly improve the granularity of the understanding of mtDNA homeostasis.


Motor deficits are common after stroke and are a major contributor to stroke-related disability and the potential for long-lasting neurobiological consequences of stroke remains unresolved. There are only a few treatments available for the improvement of motor function in stroke patients. However, the mechanisms underlying stroke recovery remain poorly understood, and effective neurorehabilitation interventions remain insufficiently proven for widespread implementation.Herein, we propose to enhance the effects of brain plasticity using a powerful noninvasive technique for brain modulation consisting of navigated transcranial magnetic stimulation (TMS) priming with transcranial direct current stimulation (tDCS) in combination with motor-training-like constraint-induced movement therapy (CIMT). Our hypothesis is that navigated low-frequency rTMS stimulus priming with precise location provided by neuronavigation on the healthy side of the brain and with anodal tDCS on the affected side combined with CIMT will induce a greater motor function improvement than that obtained with sham tDCS combined with CIMT alone. We predict that the application of this technique will result in a large reduction in cortical excitability and dis-inhibition in the affected hemisphere and lead to improvements in behavioral measures of hand function in stroke patients.The proposed study, therefore, is important for several reasons. The results could potentially lead to improved stroke therapeutics, and the approach makes use of 2 potential pathways to modulate brain function.https://clinicaltrials.gov/ct2/show/NCT04646577).This study protocol was registered in Clinical Trials Registry (The study has been reviewed and approved by the Human Research Ethics Committee of the King Fahad Specialist Hospital Dammam. The results will be actively disseminated through peer-reviewed journals, conference presentations, social media, broadcast media, print media, the internet and various community/stakeholder engagement activities. It is not only the high mortality; 50% survivors suffer from chronic disability. Thus, stroke has serious economic and public health significance. It is estimated that stroke cost 73.7 billion dollars in 2010 in United States and is projected to be 1.52 trillion dollars in 2050 (in 2005 dollars). Weakness, caused by damage of cortical networks for movement and their projections and disuse-related mechanisms, is considered a major cause of disability. Impaired function is experienced in the upper limb of two third of stroke survivors. High blood pressure, the major risk factor for stroke, is very common in Kingdom of Saudi Arabia (KSA). Incidence of stroke in Saudi population is 29.9 per 100,000. It is the third leading cause of death and in many surviving patients; it is the devastating endpoint of cerebrovascular disease. The impact of acquired brain injuries, for example stroke, upon individuals, families, and society continues to increase due to both the aging of the general population and the increasing length of post-insult survival. Stroke-related neurologic deficits, including motor function deficits, are often persistent, exerting a negative effect on the patient's quality of life.Stroke is a primary cause of death and disability globally. It is caused by an interrupted blood flow due to thrombosis or embolism to the brain which results in loss of vision, sudden paralysis, and impaired speech. In less than 15% cases, stroke leads to hemorrhage or cardiac arrest. According to the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD), stroke was responsible for 5.5 million deaths after ischemic heart disease globally (1). In United States, stroke occurs once every 40 s and someone dies every 4 minutes, with death rate estimated to 41.6% in 2007.1.2,6 It happens within many levels, from molecules to cortical reorganization. There are multiple factors that influence the outcome of stroke, including the time after stroke, the lesion's location and the integrity of cortico-spinal tracts, and the cortical and subcortical connections. In general, each hemisphere undergoes neuroplasticity changes by means of regeneration, for example, axonal and dendritic sprouting with or without reorganization of cortical motor areas, for example, modulation of the synaptic plasticity or remapping of the functional representations by diminution of GABAergic inhibition, and increased NMDA receptor binding from lessoned areas onto the unaffected ipsilesional areas which surround the lesion or the homologous areas which are within the contralesional unaffected hemisphere.,8Brain plasticity is the ability of the brain to adapt to experiences, environmental pressure, and challenges which include brain damage.The latest literature specifies that noninvasive brain stimulation (NIBS) techniques  and transcranial direct current stimulation (tDCS)) can enhance the recovery of motor functions in chronic stroke patients. Two potential roles have been described for rTMS and transcranial direct current stimulation (tDCS) in stroke recovery:(1)to inhibit, that is, downregulate, the healthy hemisphere, or(2)to enhance excitability of the lesioned hemisphere. For rehabilitation, new measures should be adopted to facilitate plasticity of brain which help in changes to occur more rapidly and more completely. Since plasticity in the hemisphere with stroke lesions leads to good recovery, one therapeutic approach such as brain stimulation should be focused to enhance brain plasticity in these areas.Downregulation of the contralesional hemisphere can lead to improved motor performance in the affected limb, thought to be through a reduction in interhemispheric inhibition to the lesioned side. Most of the spontaneous stroke recovery after the acute phase entails plastic changes within the brain.1.3–10 This kind of therapeutic approach solely relies on the functional and physiological status of each hemisphere and its balance between 2 hemispheres, which imply the importance of this physiological measure on planning an effective treatment strategy.The progress in technologies which enable non-invasive exploration of the human brain have expanded our understanding of the reorganization of the brain after ischemic stroke.Cortical physiology could be measured by TMS using paired-pulse paradigm.(1)short interval intracortical inhibition (SICI) is accepted to be GABAA-dependent since GABAA agonists increase SICI.(2)long interval intracortical inhibition (LICI) is moderated by a long lasting GABAB-dependent IPSPs and the enabling of pre-synaptic GABAB receptors on inhibitory interneurons.(3)Intracortical facilitation results from the net facilitation of mechanisms which are inhibitory and excitatory mediated by GABAA and NMDA receptors, respectively.(4)Glutamatergic cortical interneurons could be involved in intra-cortical facilitation given that it is reduced by NMDA antagonists, for example, dextromethorphan. Those measurements will enable us to shed light on how the new therapeutic approach had effect in terms of inhibition and excitation balance in each and between hemisphere.–13 which mostly has an impact on future design and choice of rehabilitation methods for each individual patient.Diffusion tensor imaging tractography (DTI) is a new technique which enables non-invasive visualization of the human brain's fiber tracts in vivoMeasurement of the corticospinal tract volume, fractional anisotropy, mean diffusivity, axial diffusivity and radial diffusivity will give us insight about inter-individual variability of the efficacy of the therapeutic intervention.,16 many of those successfully treated so far have been chronic patients that experienced stroke at least 1 year before starting CIMT therapy. It is approximated that patients whom are responsive to substantial improvement resulting from CIMT represent at least 50% of the stroke population in total current data advocate that CIMT therapy enhances motor function within a 2week period.Constraint-induced movement therapy (CIMT) is an intervention which has been used, for the most part, for treating upper extremities of stroke patients, which is based on perilesional reorganization.The effects of the treatment remain stable for many months after the end of therapy, and everyday lives of patients are adjusted accordingly with treatment effects.Recently in neuroscience, NIBS techniques like (TMS or tDCS) have shown the potential to investigate causal brain-behavior relationships and for the rehabilitation of many diseases. It is important to mention that advances in neuroscience mostly depend on the union of evidence from multiple methods. Given that every technique has limitations of its own, combining various approaches will be, theoretically, a clear advantage.1.4 Also cortical mapping showed a large-scale surfacing of new hand representations within peri-infarct motor cortex, mainly within cortical tissue underlying the stimulating electrode. Second, a recent study showed that intensive rehabilitation therapy in combination with invasive epidural electrode has a significant improvement in motor function as compared with rehabilitation alone. Third, a recent study combining brain stimulation (rTMS) and motor learning  showed that active motor cortex stimulation and motor training resulted in a significantly larger increase in motor cortex excitability in the affected hemisphere and enhanced motor performance accuracy as compared with motor training and sham stimulation. Therefore evidence supports that brain stimulation enhances the behavioral and neurophysiological effects of motor training in motor recovery.As we know coupling cortical stimulation with motor learning might enhance motor recovery: As aforementioned, both strategies for enhancing motor recovery – motor learning and cortical stimulation - are associated with similar mechanisms of action – that is, increasing local excitability within the lesioned motor cortical area (possibly through synaptic strengthening) and decreasing it in the healthy contralateral hemisphere. Therefore we hypothesized that combining both therapies will enhance its individual effect on motor recovery. Indeed a few animal and human studies support such hypothesis. First, in an elegant study, Plautz et al noted that monkeys which were submitted to electrical stimulation which was subthreshold in combination with rehabilitative training multiple months after spontaneous recovery had improvements which were significant following therapy in motor performance, which persisted for several months.There is sufficient evidence that NIBS induce neuroplastic changes which are mirrored in behavioral changes that can be observed. Albeit, the precise mechanism of NIBS in producing this neuromodulation in not well understood. Therefore, recent efforts to merge NIBS with neuroimaging in experimental paradigms were made to give a more methodical characterization of neuroplastic modulation by NIBS by way of using brain network analysis techniques.1.5,22Genetic polymorphism is also a contributing factor to affect the brain's response to disease and injury. Brain-derived neurotrophic factor (BDNF) is involved in the modulation of synaptic plasticity which is activity-dependent in the human motor cortex. A common single nucleotide polymorphism  in BDNF gene, has been associated with a decrease in the secretion of BDNF which then decreases the activity-related cortical plasticity following healthy individuals’ motor training and is associated with more errors and deficient retention in short-term motor learning.We therefore hypothesize that neuronavigation 1 Hz rTMS on health side, which provides real-time feedback and allows stimulation only when trajectory, coil position and coil rotation are within consistent, pre-determined boundaries, will be a more potent effect on the excitability of specific local cortical networks , prime with tDCS on affected side will enhance the effects of CIMT on motor function recovery in chronic stroke patients. Until recently the use of noninvasive brain stimulation coupled with motor learning was limited as rTMS or tDCS alone.Currently, detailed information about stroke or brain plasticity is not available in the Middle East.22.1Overall Approach, Participants, and Feasibility: We propose to study 40 patients (above 18 years) with first-time clinical ischemic or hemorrhagic cerebrovascular accident. Individuals with preexisting neurological or psychiatric conditions, those with unstable medical conditions, and those on any medication that might alter the TMS-tDCS measures will be excluded. A written consent will be obtained, and all the subjects will have full neurological and medical examination. Furthermore, detailed neuropsychological and behavioral assessment will also be performed in different domains. All participants will undergo a neurophysiologic study of local cortical reactivity, plasticity, and network dynamics using TMS-EMG following stroke. Participants will be randomized to active treatment with 1 Hz rTMS prime with real anodal or sham tDCS by means of randomization stratification. This approach guarantee that both groups have a close distribution in regards to the degree of paresis. Participants will be gathered into groups (strata) defined by the degree of paresis  and then randomized separately within each stratum according to a block randomization of 4 to receive real 1 Hz rTMS prime tDCS/CIMT and sham tDCS/CIMT. All participants will undergo brain magnetic resonance imaging (MRIs) for morphometric analysis and diffusion tensor imaging, and serial neurological and neuropsychological exams.–27We have developed a safe and reliable method combining TMS with EMG to characterize cortical reactivity, plasticity, and brain network dynamics in humans.2.2Anatomical and DTI: In order to guide the TMS, all participants will undergo a brain MRI for brain lesions that are structural to generate a high-resolution anatomical brain image In addition, to examine structural brain integrity, T1 image volumes will be examined in 2 primary ways a) Reconstruction which is surface based and cortical thickness analysis b) An analysis that is volume based of the amygdala, hippocampal formation and different subcortical structures. Furthermore, a diffusion tensor imaging study (DTI) will be acquired using a diffusion-weighted (DW)-EPI sequence. Probtracx (FSL) will be used to delineate corticospinal tracts and corpus callosal connectivity.2.3A rater blinded to the treatment will assess the effects of the interventions using the instruments described below. Subject enrollment, consent checklist, and screening logs will be used as well as an inclusion and exclusion checklist to assist in prescreening measures. To assess treatment efficacy (motor function changes), we will use the following instruments:The Jebsen Taylor Hand Function Test (JTHF) - this test was designed as a broad measure of hand function. This test has been used by our and other groups to assess the effects of tDCS on motor function in stroke the (2) Purdue Pegboard Test which is a test that is more sensitive to measuring changes in finger dexterity. To characterize motor function at baseline, we will use the following instruments: (3) Fugl Meyer Assessment of Motor Recovery: this tool is a progressive assessment which measures balance, motor recovery, sensation and a few joint functions in individuals with hemiparesis. Also, it includes elements which address speed and coordination (4) Modified Barthel Index Score (Activities of Daily Living): this tool assess the disability degree in each individual. It records the independence indicators in regards to the disability caused by impairments. The score ranges from 0 to 20 and it is mainly concerned with physical aspects of disability. Finally, we will use (5) the Modified Ashworth Scale: this instrument is a 6-point rating scale that is used to measure muscle tone. In addition, we will use the visual analog scale for anxiety and the visual analog scale for Pain / Comfort. Finally to measure the adverse effects of the interventions of this study, we will use (6) the Mini Mental State Examination (MMSE): is a short screening tool go assess cognitive abilities. We will use it as a quick screening method to assess any cognitive alterations that could have occurred from before or after treatment or even at follow up. The consistent scores of MMSE can suggest that the patient had no changes in cognition throughout the intervention period, which could have affected the test performance or the continuation of the program. (7) Adverse event monitoring and reporting: At each session, the patient will be asked using open-ended questions about adverse events and (8) Transcranial Magnetic/ Direct Current Stimulation Adverse Effects Questionnaire: at each session, a questionnaire will be filled by the subject to assess potential common adverse effects of TMS/tDCS  on a 5-point scale.(1) These instruments will be performed at the following time points by trained staff blinded to group assignment: during the week before the beginning of treatment (2) During day 5 and day 10 of treatment and (3) at 2 weeks of follow up, 4 weeks and 6 months after the end of treatment (Table 2.4Navigated Brain Stimulation (NBS) system  will be used to deliver TMS by a figure-of-eight (F8) coil coupled with a Magpro stimulator . Navigated TMS has the ability to online monitor the targeted cortical region, coil position, and orientation within and across sessions by using structural MRI. It delivers trial-by-trial information of an induced electric field for each TMS pulse and the localization of the induced current maxima. Data obtained by this system are reproducible, which is an essential aspect of this type of study. The display of induced currents and calculation will assist to overcome the constraints of TMS by targeting relevant structures of brain alone. As differences between different individuals in structure–function relationships, may change the induced currents and precision of the neuronavigation system, particularly in individuals with brain pathology.The repetitive TMS paradigm will be administered at 1 Hz frequency and consist of 2000 pulses delivered over a 30 minute period at 90% of the patient's motor threshold. These parameters are known to cause brain activity inhibition. The optimal stimulation site , active and resting threshold will be established in the unaffected and affected hemispheres. Assessment of EMG signals will be done at a rate of 5000 Hz. Cortical silent period (cSP) and resting and active motor threshold  will be analyzed according to the IFCN guidelines.Transcranial direct current stimulation (tDCS) individuals participating were seated in a comfortable chair. The use of the StarStim NE noninvasive wireless tDCS/EEG neurotransmitter  was used to deliver the direct current sequentially and record the EEG data. A placement of small Ag/AgCl gelled electrodes was done over the lesioned hemisphere  and area F3 (return electrode), with a surface contact area of 3.14 cm2 specific to the StarStimNE device . A control box device, which is wirelessly connected to a computer and communicated with the NIC , will be connected to the electrodes. throughout anodal stimulation, delivery of direct current took place from a current-control circuit at an intensity of 1.5 mA and maintained for 20 minutes. As for the sham stimulation, placement of the electrodes was done in the same position and individuals participating were given a short ramp up  at the start and end of the stimulation period.Assessment of cortical excitability – single and paired pulse TMS We will investigate changes in cortical excitability evaluating the motor evoked potential and the resting motor threshold  of CIMT  is conservative to detect a clinically meaningful difference between groups (active 1 Hz rTMS prime with tDCS/CIMT and sham tDCS/CIMT). We will assume a type I error of 5% , a type 2 error of 10% (beta), therefore, the power is going to be 90%. A t-test will be used ; where 15 individuals per arm will be needed . We expect a 20% rate of loss to follow up or drop out. Moreover, we will assume that individuals who drop out will not improve from the last measured point; therefore, the sample size will increase by 20% to 36 participants (18 per arm). Furthermore, in case of unexpected factors such as higher placebo response, we increased the sample size further to 40 patients.PROTECTION OF HUMAN SUBJECTS This study aims to enroll adult patients with a history of cerebral infarct that is chronic and associated with stable and significant motor deficits. Both ischemic and hemorrhagic stroke subtypes will be included. The entry criteria for patient enrollment are the following:(1)age 18 to 90;(2)first-time clinical ischemic or hemorrhagic cerebrovascular accident as noted in the radiological (or physician's) report;(3)the ability to extend =20° at the wrist and 10° at the metacarpophalangeal and interphalangeal joints of all digits;(4)participants must demonstrate adequate balance while wearing the restraint;(5)the ability to stand from a sitting position and the ability to stand for at least 2 minutes with or without upper extremity support;(6)weakness, defined as score of 15 to 55 (out of 66) on arm motor Fugl-Meyer scale; and(7)stroke onset more then 6 months prior to study enrollment.Exclusion criteria:(1)having significant disability before stroke;(2)any history of depression before the stroke (where appropriate);(3)Any considerable decline in language reception, alertness or attention that might hinder with understanding the instructions for the motor testing;(4)excessive pain in any joint of the paretic extremity;(5)contraindications to single-pulse TMS  such as metal head implants;(6)advanced cardiac, pulmonary, kidney or liver disease;(7)a terminal medical diagnosis consistent with survival 1 year;(8)coexistent major neurological or psychiatric disease (to decrease the number of confounders);(9)a history of significant drug abuse in the prior 6 months;(10)the use of certain neuropsychotropic drugs such as tricyclics, antidepressants, or 51 of 66 carbamazepine;(11)active enrollment in a separate intervention study targeting stroke recovery;(12)previously applied constraint-induced motor therapy and/or tDCS treatment for stroke; and(13)a history of epilepsy before stroke (or episodes of seizures within the last six months).2.6The study has been reviewed and approved by the Human Research Ethics Committee of the King Fahad Specialist Hospital Dammam (NEU329). The results will be distributed actively through journals which are peer-reviewed, conference presentations, broadcast, social and print media, the internet and multiple community/stakeholder engagement activities.3Our proposed project is designed to challenge the current research and shift it with the clinical practice ideals; therefore, revise the current clinical practice. We believe that our hypothesis contributes an innovative account of the different findings noted in stroke rehabilitation and a practical approach to understand the fundamental features of motor decline. Also, we believe that identifying and understanding the regulation and consequences of inefficient plasticity may enable novel plasticity-based therapeutic interventions in the future. Moreover, our approach can help in inspecting the homeostatic plasticity at the level of brain networks that is induced by NIBS; it is a vital phenomenon for the of application of NIBS in managing diseases that involve pathologically altered cortical excitability, such as stroke. If we obtain our proposed objectives, this research will allow for a transformation of the process of brain plasticity through developing evidence-based technologies and interventions directed at(1)establishing markers for altered cortical plasticity and(2)the modulation of plasticity to improve motor performance.4In summary, such combined approaches to studying and quantifying the neurophysiological processes associated with neuroplasticity are critical to help identify, monitor, and potentiate neuroplasticity, which is crucial for functional recovery in patients suffering from brain lesions such as stroke.We thank Mr. Brent for technical assistance.All listed authors developed different substantial activities. FA, WKY and SB design the experiment and generate hypothesis. MI, AN and NE were involved in drafting the manuscript. Each author participated sufficiently in writing and reviewing the manuscript. All authors read and approvedConceptualization: Fawaz Al-Hussain, Woo-Kyoung Yoo, Shahid Bashir.Data curation: Fawaz Al-Hussain, Eman Nasim, Muhammad Iqbal, Niaz Asim, Shahid Bashir.Investigation: Fawaz Al-Hussain, Eman Nasim, Muhammad Iqbal, Shahid Bashir.Methodology: Fawaz Al-Hussain, Muhammad Iqbal, Nouf Altwaijri, Niaz Asim, Shahid Bashir.Writing – original draft: Fawaz Al-Hussain, Eman Nasim, Muhammad Iqbal, Nouf Altwaijri, Niaz Asim, Woo-Kyoung Yoo, Shahid Bashir.Writing – review & editing: Fawaz Al-Hussain, Eman Nasim, Muhammad Iqbal, Nouf Altwaijri, Niaz Asim, Woo-Kyoung Yoo, Shahid Bashir.