Assessment of Bidirectional Relationship between Polycystic Ovary Syndrome and Depression: A Two-Sample Mendelian Randomization Study

Background : Evidence from observation studies has implied an association between polycystic ovary syndrome (PCOS) and risk of depression. Nevertheless, it remains elusive if the identified correlation is causal or owing to biases in observation researches. Hence, we utilized a bidirectional two-sample Mendelian randomization (MR) method to evaluate the potential causal relationship between PCOS and depression. Methods : Genetic instruments for PCOS and depression were acquired from two large genome-wide association studies (GWASs). MR analyses were completed via the inverse-variance weighted (IVW) method and weighted median approaches. The underlying pleiotropy was tested by MR-Egger regression, and leave-one-out method was used to evaluate the stability of MR results. Results : Using the IVW analyses (odds ratio (OR) = 1.07, 95% confidence interval (CI) = 1.01–1.06, p < 0.01) and weighted median approach (OR = 1.04, 95% CI = 1.00–1.08, p < 0.05), we found that PCOS was related to an elevated risk of depression. MR-Egger regression did not identify potential horizontal pleiotropy. Sensitivity analyses using leave-one-out method also provided supportive evidence. In the reverse MR analyses, we did not observe causal effect of depression on PCOS ( p > 0.05). Conclusions : The present study provides evidence to support a potential causal association between PCOS and an elevated risk of depression. Hence, early psychological intervention for PCOS might show anti-depression benefits.


Introduction
Polycystic ovary syndrome (PCOS) is a commonly seen endocrine disturbance which is the major cause of anovulatory infertility and affects up to 15% of women of reproduction age.It is characterized by androgenism, ovulatory dysfunction, obesity, menstrual dysfunction, metabolic and psychiatric abnormalities [1,2].Hyperandrogenemia is present in 15%-45% of patients with PCOS [3], such as acne, hairiness, and increased level of free testosterone in peripheral blood.Moreover, it is known that women with PCOS are more likely to suffer from psychological problems, especially anxiety and depression [4].
The pathogenesis of PCOS remains unclear.Clinical observations and animal experiment data suggest the assumption that PCOS is inherited and induced by developmental programming of normal genetic mutations.Those genes would be magnified by exposing to in-utero androgen and stimulated by various post-natal life-style and environment factors.Chemistry substances that disrupt endocrine harbor the possibility to affect the developmental programming of PCOS susceptible genes [5].
Depression is a more and more commonly seen disease which constitutes remarkable health-care challenge.In 2008, WHO considered severe depression the 3rd cause of disease burden across the globe and forecasted that the problem will rank first by 2030 [6].Depression influences females more than males [7] and is the most commonly seen psychological issue for females with PCOS.Females with PCOS present an 8-fold greater incidence of depression in contrast to females with no PCOS [8].The different pathophysiologic causal links inducing depression are insulin resistance (IR), disturbances in the hypothalamic pituitary adrenal (HPA) axis and hyperandrogenism [2].
Although previous studies provided a suggestive link between PCOS and the risk of depression, residual confounding and reverse causation are difficult to eliminate in observational studies.Recently, there is an alternative method to investigate the potential causal association unbiasedly, two-sample Mendelian randomization, which depends on gene mutations as instrumental variables (IVs) to assess the causal association between an exposure and a result [9].After random location in the process of meiosis, single-nucleotide polymorphisms (SNPs) used as in- strumental variables (IVs) remains stable and non-modified throughout a life-time of environment exposure, which makes them independent of confounding factors or reverse causation [10].
Based on gene mutations, Mendelian randomization (MR)-offered proofs of PCOS-related causality pertaining to several physical disorders have been found [11].Moreover, previous Mendelian randomized study also explored the causal associations between PCOS and psychiatrical disorder and discovered that PCOS increased the risk of obsessive-compulsive disorder, but was not associated with the other four psychiatric disorders (anxiety disorders, bipolar disorders, severe depression disorders, or schizophrenia) [12].On the basis of the release of a new database with a larger sample size, we conducted this updated bidirectional Mendelian randomized analyses to assess whether the underlying causality between PCOS and depression could have different outcomes.

Data Source and Study Samples of PCOS and Depression
The genome-wide association study (GWAS) dataset of PCOS was retrieved from the European ancestry.Using a meta-analysis technique, an adequate sample size from seven original datasets was obtained (10,074 cases and 103,164 controls) [13], which yielded better statistical power in identifying the associated SNPs.The diagnosis criteria of PCOS were on the foundation of the NIH or Rotterdam standards, or self-reporting in different original datasets.More specific information regarding the cohorts, genotyping, quality control, and imputation can be viewed in previous studies [13].
The gene correlation estimates of depression were acquired from a meta-analysis [14].It enrolled a total of 807,553 participants (246,363 cases and 561,190 controls) from three cohorts: 23andMe [15] (75,607 cases & 231,747 controls), UK Biobank [16] (127,552 cases & 233,763 controls), and PGC_139k [17] (43,204 cases & 95,680 controls), after excluding overlapping samples.All the samples were of European descent.In the 23andMe cohort, individuals with depression were defined via self-reporting using on-line investigation.Detailed description of these questions can be accessed in the publication by Hyde CL et al. [15].In the UK Biobank cohort, depression was defined according to the self-report using a touchscreen survey.Participants with bipolar disorder, schizophrenia or personality disorder were excluded.Additionally, the enrolled samples were strictly confined to the "White British" to reduce the population architecture bias.The PGC_139k cohort used samples from the earlier released data of 23andMe and UK Biobank cohorts.The overlapping samples were removed from the combined cohort.

Genetic Instruments Selection
The genetic instruments of PCOS and depression were identified with a genome-wide statistical threshold of p < 5 × 10 −8 .By further calculating the linkage disequilibrium (LD) of related SNPs, independent SNPs (LD r 2 <0.001, kb <1 Mb) were retained.Additionally, we also selected steiger-MR approach [18] to verify if the SNPs elucidated remarkably more variance in exposure in contrast to result.The opposite might reveal reverse causation and violate the basic MR assumptions.In addition, the strength of gene instruments was assessed via F-statistics to avoid the weak instrument variables bias.F-statistics were calculated via the formula below: F-statistics = (Beta/Se) 2 , and its mean was regarded as the overall statistics.F-statistics >10 indicates strong statistical power [19].Eventually, a total of 13 and 50 SNPs were selected as instrument variables for PCOS and depression, respectively.Detailed information of the IVs for PCOS and depression is summarized in Tables 1,2, separately.

Statistical Analyses
Three methods including the inverse variance weighted (IVW), weighted median value, and MR Egger regression were utilized to assess the bilateral causal association between PCOS and depression (Fig. 1).The IVW approach hypothesizes that all the IVs are effective.It combines the effects of IVs and then yields an overall weighted effect.The weighted median estimator [20] can produce stable causality estimates even when 50% IVs are not valid.Two methods were undertaken to identify potential pleiotropy.First, we applied MR Pleiotropy Residual Sum and Outlier (MR-PRESSO) method to identify potential pleiotropic outliers, and MR-PRESSO conducts a global test of heterogeneity to identify potential horizontal pleiotropy [21].Then, the intercept test from MR-Egger [22] was also applied to assess the directional pleiotropy.The intercept term significantly away from zero in statistics indicates the presence of pleiotropy and violation of basic MR assumptions.
Leave-one-out (LOO) analysis was used to identify the potential influential SNPs in the causality estimates between PCOS and depression.p < 0.05 (two-sided) was set as the threshold of statistical significance.The entire analytical process and figures were made via the R software (version 3.6.5)(https://www.r-project.org/).

Genetically Predicted PCOS on Depression
As shown in Fig. 2, genetically predicted PCOS was associated with a 1.04-fold increased risk of depression by the IVW method (95% confidence interval (CI) = 1.01-1.06,p = 0.003).This increased risk was also replicated by the weighted median approach (OR = 1.04, 95% CI = 1.00-1.08,p = 0.03), suggesting a potential risky role of PCOS in the suffering of depression.No pleiotropic signs (MR-Egger intercept = -0.004,p = 0.733; MR-PRESSO global test p = 0.179) were observed (Table 3).As displayed in Fig. 3A, with the increase of the SNP effect on PCOS, the SNP effect on depression increased as well.The LOO analyses disclosed that no influential SNP existed in the PCOS-depression causal association.

Genetically Predicted Depression on PCOS
The estimates of genetically forecasted depression on PCOS were displayed in Fig. 2. In the IVW analyses, the OR was 1.07 (95% CI = 0.76-1.50,p = 0.706), which did not suggest a risky role of depression in the occurrence of PCOS.The estimate from the weighted-median method (OR = 0.90, 95% CI = 0.55-1.47)was also insignificant (p = 0.674).There were no signs of pleiotropy (MR-Egger intercept = -0.003,p = 0.961; MR-PRESSO global test p = 0.319) in Table 3.
The scatter plot visualizing the SNPs-depression association against SNPs-PCOS association is displayed in Fig. 3B.The LOO analyses indicated that no influential SNP existed in the depression-PCOS association.

Discussion
Herein, our team completed a two-sample MR and identified a potential causal association of PCOS with depression via the biggest GWAS data set to date.This study suggested that PCOS might increase the risk of depression.In contrast, we did not find evidence that depression may increase the risk of PCOS.
Previously, observational researches have unveiled the underlying correlation between depression and PCOS.Açmaz et al. [23] discovered that PCOS group (n = 86) with infertility had higher depression scores.Altinkaya et al. [24] found that Beck Depression Inventory scoring was remarkably greater in patients with PCOS (n = 83) in contrast to normal controls [24].An Iranian case control study found that there existed a remarkable diversity between PCOS (n = 742) and controls (n = 798) in depression (18.9% vs. 7.9%; p < 0.001) [25].A cross-sectional study showed that females with PCOS displayed severer anxiety (p = 0.007) and depression (p = 0.048) in contrast to females without PCOS [26].A study in Southwest China involved 120 out-patients with PCOS and 100 normal controls showed that the prevalence of depression (27.5% vs. 3.0%) was greater in PCOS sufferers in contrast to controls (p < 0.05) [27].Coherent with those aforesaid observation researches, data from this study herein backed the assumption that PCOS might be related to an elevated risk of depression.
Despite the fact that the biofunction of PCOS in the depression progression remains elusive, some researches have offered reasonable elucidation in this regard, one of which is hyperandrogenism [28,29].Second, a positive association between insulin resistance and depression was found [30,31].The randomized control trial (RCT) of Greenwood et al. [32] (738 PCOS females) potently reveals that insulin resistance is a causation factor for PCOS-related depression.In addition, PCOS is considered a proinflammation status featured by elevated contents of proinflammation biomarkers.Hence, there exists a probability of an inflammation association between depression and PCOS [33].It is probable that the inflammation biomarkers in PCOS can cross the blood-brain barrier, inducing the progression of depression [33].While the above theory-wise elucidation is reasonable, more researches are needed to reveal the potential causal link between PCOS and depression.
The major contribution of the present research is the utilization of the MR method, which has been extensively utilized to explore the causality of PCOS with the risks of other diseases.In addition, our team obtained the SNPs of depression and PCOS via the biggest GWAS datasets to date.The SNPs herein were remarkably related to PCOS at genome-wide significance, hence decreasing potential breach of the first hypothesis of MR.In addition, the Fstatistics for the IVs all satisfied the liminal value of Fstatistics >10, revealing that the analyses were not likely to be influenced by weak instrument bias [34].The MR-Egger regression analyses were completed to study the data and did not observe the existence of directional pleiotropy [22].Those coherent outcomes revealed the stability of the discoveries in the present paper.
Nonetheless, there are certain limitations of our research.Firstly, as our analyses were limited by European individuals, the results might not be extended to other races.Nevertheless, this remarkably decreased the underlying influences of population stratification bias as well.Secondly, while the MR method may offer a non-biased outcome because of the diminished confounders, the gene-milieu and gene-gene interplay might influence the progression of depression or PCOS inevitably.Thirdly, the MR analyses of PCOS and depression were on the foundation of summary statistics with comparatively smaller sample size, and the underlying side effects of PCOS on the risks of depression ought to be further investigated in bigger sample size.In addition, as the related data of all PCOS phenotypes were unavailable, our team merely investigated the correlation between PCOS and depression, and our team did not stratify the outcomes herein as per the diverse PCOS phenotypes.More researches highlighting the correlation of PCOS with depression are still needed.

Conclusions
To sum up, the present research offered evidence to suggest potential causality between PCOS and an elevated risk of depression amongst European individuals.Nevertheless, the accurate roles and the potential biology processes of PCOS in the progression of depression require deeper explorations.

Fig. 3 .
Fig. 3.The scatter plot for bidirectional Mendelian randomization.The subplots (A) represents the causal effects of PCOS on depression; The subplots (B) represents the causal effects of depression on PCOS.