Vitamin D deficiency and high serum IL-6 concentration as risk factors for tubal factor infertility in Chinese women

Nutrition 49 (2018) 24–31

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Nutrition j o u r n a l h o m e p a g e : w w w. n u t r i t i o n j r n l . c o m

Applied nutritional investigation

Vitamin D deficiency and high serum IL-6 concentration as risk factors for tubal factor infertility in Chinese women Weiwei Chen M.Med. a,1, Xianting Jiao M.Med. b,1, Jun Zhang Ph.D., M.D. b, Lei Wang M.Med. b, Xiaodan Yu Ph.D., M.D. a,* a

Department of Developmental and Behavioral Pediatrics, Shanghai Children’s Medical Center affiliated to Shanghai Jiaotong University School of Medicine, Ministry of Education-Shanghai Key Laboratory of Children’s Environmental Health, Shanghai, China b MOE-Shanghai Key Lab of Children’s Environmental Health, Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

A R T I C L E

I N F O

Article history: Received 20 September 2017 Received in revised form 21 November 2017 Accepted 27 November 2017 Keywords: 25-hydroxyvitamin-D Female infertility Tubal factor infertility Inflammatory cytokines Interleukin 6

A B S T R A C T

Objectives: The aim of this study was to investigate the relationship between 25-hydroxyvitamin-D [25(OH)D] and female infertility and to further explore the role of inflammatory cytokines. Method: We recruited 356 infertile women diagnosed with tubal factor infertility (TFI) or polycystic ovary syndrome (PCOS) or endometriosis, as well as 180 fertile women. Serum concentrations of 25(OH)D, interleukin (IL)-6, IL-1 β, and interferon-α were measured. Results: The 25(OH)D concentration in TFI women was the lowest (16.9 ng/mL) and was significantly different from that in the fertile women (19.4 ng/mL; P < 0.05)]; whereas women with TFI had higher IL-6 concentrations. After adjusting for confounders, 25(OH)D deficiency presented a risk factor for TFI (odds ratio [OR], 4.2; 95% confidence interval [CI], 1.5–11.3). There was a dose–effect relation between IL-6 tertiles and TFI: the higher the IL-6, the higher the risk for TFI (middle versus low: OR, 3.7; 95% CI, 1.5–9.5; high versus low: OR, 13.2; 95% CI, 4.8–36.4). IL-6 showed a negative correlation with 25(OH)D (r = −0.19). Women with both high IL-6 and low 25(OH)D had the highest risk for TFI (OR, 10.6; 95% CI, 4.2–26.3). Conclusions: Both vitamin D deficiency and high serum IL-6 concentration are risk factors for TFI. Serum 25(OH)D concentration was significantly and negatively correlated with serum IL-6. There was an interaction between IL-6 and 25(OH)D for the risk for TFI-related infertility. We hypothesized that vitamin D might reduce the risk for TFI through suppressing the production of IL-6. © 2017 Elsevier Inc. All rights reserved.

Introduction Female infertility is a critical problem for reproductive health worldwide, with a reported prevalence of 1.2% to 3.1% in childseeking women [1] and 15% in couples [2]. The etiologies of female infertility include ovulation (15%–20%; polycystic ovary syndrome [PCOS] is the most common cause), tubal problems (15%–40%), endometriosis (5%–10%), and unexplained cases (20%–30%) [3]. Infertility can result in depression and distress, as well as ostracism and discrimination [4–6].

This study was supported by the Chinese National Natural Science Foundation (no. 81373004), the National Basic Research Program of China (973 Program) (No 2014 CB943300), and Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (No 20152220). * Corresponding author: Tel.: +86 21 250 78868; fax: +0086 21 25078875. E-mail address: [email protected] (X. Yu). 1 WC and XJ are co-first authors. https://doi.org/10.1016/j.nut.2017.11.016 0899-9007/© 2017 Elsevier Inc. All rights reserved.

It is well known that the major role of vitamin D, a steroid hormone, is related to calcium metabolism and bone structure [7]. Increasing evidence demonstrates that vitamin D also modulates reproductive processes in women. The biological actions of vitamin D are mainly mediated through vitamin D receptor [8], which is expressed in reproductive tissues, including testis, ovary, uterus, and placenta [9]. Researchers are increasingly interested in the potential role of vitamin D in the prevention of female infertility. Four experimental animal studies revealed that vitamin D–deficient female mice were infertile and exhibited uterine hypoplasia and absent corpora lutea [7,10–12]. As for humans, eight original articles have investigated the relation between serum concentrations of 25-hydroxy-vitamin D [25(OH)D] and pregnancy rates in assisted reproduction technology, but the outcomes are inconsistent. It has been proposed that women with highserum vitamin D concentrations have high chances of pregnancy, whereas vitamin D deficiency (VDD) may impair pregnancy rates in infertile women undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection [13–16]. On the contrary, an

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inverse relationship between 25(OH)D concentration in follicular fluid and IVF clinical pregnancy rates also is described [17]. Moreover, Franasiak et al. revealed that vitamin D status was unrelated to pregnancy rates in women undergoing euploid blastocyst transfer [18]. Two studies from Iran found no influence of serum 25(OH)D concentrations on the pregnancy outcome [19,20]. Meanwhile, to our knowledge, only one epidemiologic study to date has investigated the relationship between vitamin D and female infertility caused by different diseases including poor ovarian response, TFI, PCOS, and endometriosis [21]. In the present study, we observed a high rate of VDD (40.1%) among 1072 white European women who were infertile. Furthermore, 25(OH)D concentration was reported to be positively associated with endometriosis history. However, the sample size of each disease group was small (e.g., tubal factor group: n = 66; PCOS group: n = 161). There are insufficient data to accurately evaluate the effects of vitamin D on female infertility. Available literature is supportive of roles for vitamin D in fertility through the regulation of the ovarian folliculogenesis, extracellular calcium and phosphorus, hypothalamus–pituitary axis, and uterine implantation [22,23]. However, additional vitamin D–mediated mechanisms responsible for the prevention of infertility are still waiting to be explored. As is well known, inflammation plays a significant role in infertility and gynecology by affecting ovulation and hormone production [24]. Referring to previous literature, we discovered that proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1 β, IL-6, IL-8, and IL-10 are aberrantly expressed in the peritoneal fluid and plasma of women with endometriosis [25–28]. The most significant cause of inflammatory infertility is Chlamydia trachomatis infection, which may result in infertility in 10% to 30% of infertile couples in developed countries [24]. The induction of proinflammatory cytokines after infection can damage the epithelium of the fallopian tubes, causing loss of function [29]. Increasing evidence has linked VDD to various inflammatory diseases such as atherosclerosis-related cardiovascular disease, asthma, inflammatory bowel disease, chronic kidney disease, and nonalcoholic fatty liver disease [30–32]. Vitamin D exhibits antiinflammatory actions that may contribute to its beneficial effects in several cancers [33]. An in vitro study observed that vitamin D dose-dependently suppresses lipopolysaccharide-induced IL-6 and TNF-α production by human monocytes [34]. The only epidemiologic study presented that women with sufficient vitamin D had lower odds ratios for periodontitis via an antiinflammatory transcriptional mechanism (IL-6, IL-1 β, B lymphocyte chemoattractant, etc.) [35]. In summary, it has been demonstrated that proinflammatory cytokines such as interferon-α (IFN-α), IL-1 β, IL-6 are related to antiinflammatory function of vitamin D. Considering the above conclusions, we conducted this multicenter study to investigate the correlations among 25(OH)D, inflammatory cytokines, and infertility. The objectives of the present study were to validate the relationship between 25(OH)D and female infertility, and to further explore the role of the inflammatory cytokines. Material and methods Study population This study was based on a case–control approach assessing the relationships between 25(OH)D, inflammatory cytokines, and female infertility in China. Women 20 to 40 y of age who presented to the fertility clinics of three cooperative hospitals (Women’s Hospital Affiliated to Zhejiang University School of

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Medicine, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, and Reproductive Hospital Affiliated to Shandong University) for treatment of infertility from 2013 to 2015 were considered eligible for the study. Except for infertility, none of the patients at enrollment had history or had been cured of other serious underlying diseases such as cancer; cerebrovascular disease; trauma; liver, kidney, heart, or respiratory diseases; or chromosome abnormalities. According to World Health Organization criteria, infertility was defined as not conceiving a pregnancy after ≥12 mo of unprotected intercourse regardless of whether or not a pregnancy ultimately occurred [36]. We selected 377 women who were diagnosed with infertility. This group was further divided into three subgroups based on the etiology (PCOS-, TFI-, or endometriosis-related). The diagnoses of PCOS and TFI were confirmed through participant-provided medical letters and reports from previous medical investigations. The diagnosis of endometriosis was confirmed by surgical visualization using laparoscopy. In all, 204 women without reproductive disorders seeking infertility treatment because of male reproductive dysfunction during this period were selected as controls. There were no specific matching criteria for controls. Twenty-one cases and 24 controls failed to provide blood samples or complete data, leaving 536 women (356 cases and 180 controls) for final analysis. None of the participants had hysterectomy at time of recruitment. Written informed consent was obtained from each participant. The study protocol was approved by the Medical Ethics Committee of Shanghai Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and three collaborative hospitals.

Data collection A face-to-face interview with participants was conducted by a trained interviewer using a standardized questionnaire to collect a range of information, including demographic factors (age, occupation, education, and family incomes), menstrual and reproductive history (age at menarche, history of pregnancy, menopausal status, and gynecologic surgeries), survey month, vitamin D supplementation in 30 d, sun exposure, family history, and lifestyle behaviors (including current alcohol consumption and smoking). Medical information such as anthropometric variables (height and weight), history of contraception, gynecologic examination, and chromosome analysis was obtained from medical records. A 10-mL blood sample was obtained from each participant and centrifuged for separation of serum within 1 h of collection and stored at −80°C until analysis.

Serum 25(OH)D and three inflammatory cytokine measurements Measurement of serum 25(OH)D was performed by radioimmunoassay (RIA) according to the manufacturer’s instructions [25(OH)Vitamin D total-RIA-CT, DIAsource ImmunoAssays, Nivelles, Belgium]. The intra- and interassay coefficients of variations (CV) were <6% and <10%, respectively. Vitamin D deficiency was defined as a 25(OH)D concentration of <20 ng/mL [37]. Serum concentrations of three inflammatory cytokines (IL-6, IFN-α, and IL-1 β) were measured by cytometric bead array, according to the manufacture’s protocol (BD Biosciences, San Jose, CA, USA). The analytical method was described in detail previously [38]. Both intra- and interassays CV were <10%.

Statistical analysis The primary outcomes of the present study were the prevalence of VDD in infertile women in China and the contribution of VDD to the risk for female infertility. The sample size was calculated according to the following formula: Z12−α 2 p (1 − p) [39]. Because there were no data suggesting the prevalence of d2 VDD among infertile women in China, we referred to the recent reported prevalence of VDD among infertile Italian women from 2013 to 2015, which was approximately P = 46% [40]. As in the majority of studies, P values were consid-

N=

ered significant at < 0.05; hence, Z1−α = 1.96 was used in formula. We decided to calculate this sample size with the precision/absolute error (d) of 5%; then we calculated that n = 382. Finally, we recruited 536 women. Data are presented as mean ± SD for continuous variables, and as frequency or percentage for categorical variables. Percentages are rounded to whole numbers. The Wilcoxon rank-sum test and χ2 tests were used to determine any statistical difference among the four groups. We presented the distribution of 25(OH)D and three inflammatory cytokines in the cases and controls using the median and interquartile range (IQR). Differences in the serum concentrations of the above compounds were compared between cases and controls using Wilcoxon’s nonparametric test. To make clear the relationship among serum 25(OH)D concentrations, IL-6, and TFI-related infertility, we performed unconditional logistic regression to calculate the odds ratios (ORs) as estimates of the relative risk and their 95%

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W. Chen et al. / Nutrition 49 (2018) 24–31

Table 1 Characteristics of the study population by case status (N = 536)* Characteristic

Age (y) BMI (kg/m2) Age at menarche (y) Menstrual cycle (d) Menstrual regularity Regular Irregular Missing Survey month Spring (March–May) Summer (June–August) Autumn (September–November) Winter (December–February) Missing Vitamin D supplementation in 30 d No Yes Sun exposure Often Sometimes Rarely Never Missing Education Less than high school High school graduate College graduate or higher Missing Annual income (RMB/person) <30 000 30 000–50 000 50 000–100 000 ≥100 000 Missing Ever pregnant (parity) No Yes Missing Diabetes or IGT No Yes Missing

Control (n = 180) n (%) or mean (SD)

Case (n = 356) n (%) or mean (SD) PCOS (n = 225)

TFI (n = 109)

Endometriosis (n = 22)

28 (3.8) 23 (3.4) 14.8 (2.6) 30.3 (4.7)

27.9 (3.4) 25.0 (4.3)† 14.1 (1.6) NA

28.6 (3.1) 22 (3.6) 14.2 (1.7) 30.3 (3.7)

29 (3.4) 22.4 (2.4) 14.3 (1.3) NA

158 (88) 20 (11) 2 (1)

18 (8)‡ 189 (84) 18 (8)

93 (85) 12 (11) 4 (4)

13 (59)‡ 8 (36) 1 (5)

20 (11) 124 (69) 18 (10) 0 (0) 18 (10)

12 (5) 176 (78) 0 (0) 1 (0) 36 (16)

26 (24)‡ 54 (50) 21 (19) 1 (1) 7 (6)

4 (18) 15 (68) 2 (9) 0 (0) 1 (5)

173 (96) 7 (4)

207 (92) 18 (8)

85 (78)‡ 24 (22)

15 (68)‡ 7 (32)

46 (26) 30 (17) 32 (18) 31 (17) 41 (23)

68 (30) 44 (20) 49 (22) 11 (5) 53 (24)

7 (6)‡ 32 (29) 16 (15) 47 (43) 7 (6)

2 (9)‡ 4 (18) 1 (5) 13 (59) 2 (9)

101 (56) 40 (22) 34 (19) 5 (3)

117 (52) 53 (24) 49 (22) 6 (3)

65 (60) 11 (10) 27 (25) 6 (6)

10 (46)‡ 2 (9) 8 (36) 2 (9)

99 (55) 29 (16) 14 (8) 4 (2) 34 (19)

150 (67) 30 (13) 16 (7) 5 (2) 24 (11)

58 (53) 14 (13) 8 (7) 2 (2) 27 (25)

13 (59) 3 (14) 2 (9) 0 (0) 4 (18)

128 (71) 40 (22) 12 (7)

154 (68) 61 (27) 10 (4)

44 (40)‡ 58 (53) 7 (6)

11 (50)‡ 7 (32) 4 (18)

161 (89) 4 (2) 15 (8)

153 (68)‡ 61 (27) 11 (5)

97 (89) 12 (11) 0 (0)

22 (100) 0 (0) 0 (0)

BMI, body mass index; IGT, impaired glucose tolerance; PCOS, polycystic ovary syndrome; TFI, tubal factor infertility. * Values are mean (±SD) for continuous variables, and n (%) for categorical variables. † P < 0.05 vs the control group, Wilcoxon rank-sum test. ‡ P < 0.05 vs the control group, Pearson χ2 test.

confidence intervals (CIs). Considering that the exposure–disease relationship may not be linear or monotonic, 25(OH)D concentration was divided into two categories based on VDD definition: ≥ 20 and <20 ng/mL, with the former serving as the referent [37]. Additionally, we categorized the IL-6 concentration into three tertiles, with the lowest tertile serving as the referent. According to our univariate analysis and previous studies, we considered the following variables as potential confounders and adjusted for in the multivariable logistic regression: survey month, age, BMI, education, family income, sun exposure, parity, and vitamin D supplementation in 30 d. Our data showed that most women were nondrinkers and nonsmokers; thus, these two variables were not considered for adjustment. We analyzed the correlations between 25(OH)D and inflammatory cytokines by Pearson correlation analysis. Furthermore, we applied a three-piecewise linear regression model to examine the threshold effect of the 25(OH) D on IL-6 concentration according to the smoothing plot. Two inflections of 25(OH)D, at which the relationship between IL-6 and 25(OH)D concentration began to change and became eminent, were determined using a trial method. The latter was to move the trial inflection point along a predefined interval and detect the inflection point that gave the maximum model likelihood. Interaction analysis was conducted according to 25(OH)D concentration (<20 and ≥20 ng/mL) and IL-6 concentration (
www.empowerstats.com, X&Y Solutions, Inc., Boston, MA, USA). A two-sided significance level of 0.05 was used to evaluate statistical significance.

Results This case–control study recruited 356 infertile and 180 fertile women (N = 536). Sociodemographic and reproductive characteristics of the participants are summarized in Table 1. The distributions among the four subgroups were acceptably similar, except for BMI, menstrual regularity, education, pregnancy history, diabetes-related disorders, and so on. Most patients were enrolled during the month of June to August (summer; 69%); however, compared with other groups, significantly fewer patients in the TFI group (50%) were surveyed during summer. Furthermore, the frequency of sun exposure was significantly lower in the TFI and endometriosis groups. Women with PCOS or endometriosis were more likely to have menstrual, reproductive, or diabetes-related disorders than the control women. There

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Table 2 Serum concentrations of 25(OH)D and inflammatory cytokines by case status (N = 536) Concentrations

Serum 25(OH)D (ng/mL) Serum 25(OH)D† ≥20 ng/mL <20 ng/mL IL-6 (pg/mL) IFN-α (pg/mL) IL-1 β (pg/mL)

Median (IQR) or n (%) Control (n = 180)

PCOS (n = 225)

TFI (n = 109)

19.4 (15.8–22.7)

19.1 (15.9–24.1)

16.9 (14.1–19.4)*

82 (46) 98 (54) 4.6 (3.6–6) 7.0 (5.8–8.1) 2.9 (2.1–3.7)

100 (44) 125 (56) NA NA NA

Endometriosis (n = 22) 17 (13.4–18.9)

25 (23) 84 (77) 7.3 (4.7–10.3)* 6.5 (5.5–7.5) 2.5 (2–3.4)

5 (23) 17 (77) 10.0 (5.7–12.1)* 6.5 (5.7–7.7) 2.8 (2.7–3.5)

25(OH)D, 25-hydroxyvitamin-D; IFN, interferon; IL, interleukin; IQR, interquartile range; PCOS, polycystic ovary syndrome; TFI tubal factor infertility. * P < 0.05 vs the control group, Wilcoxon rank-sum test. † P < 0.05, Pearson χ2 test.

were no significant differences between groups in age, age at menarche, or household income. Table 2 shows the serum concentrations of 25(OH)D and three cytokines by case status. The median concentration of 25(OH)D was 18.5 ng/mL (IQR, 15.4–22.7 ng/mL). VDD was defined as serum 25(OH)D concentration <20 ng/mL [37] and the overall prevalence was 60%. Women with TFI had significantly lower median concentrations of 25(OH)D than the control group (P = 0.003). On the contrary, the median concentrations of IL-6 in women with TFI and endometriosis were significantly higher than the control group (P < 0.001 for each). The serum concentrations of other cytokines were comparable among the three study groups. In the crude analysis, increased odds of TFI-related infertility were observed for women who had VDD (Table 3). After adjustment for confounders, serum concentrations of 25(OH)D were inversely associated with elevated risks for TFI-related infertility (<20 versus ≥20 ng/mL: OR, 4.2; 95% CI, 1.5–11.3; P = 0.005). However, serum concentrations of IL-6 were significantly associated with TFI-related infertility (second versus lowest tertile: OR, 3.7; 95% CI, 1.5–9.5; P = 0.006; highest versus lowest tertile: OR, 13.2; 95% CI, 4.8–36.4; P = 0.0001). As we observed, there was a dose-dependent relationship between IL-6 tertiles and TFI: the higher the IL-6, the higher the risk for TFI. There were no remarkable associations between TFI-related infertility and other inflammatory cytokines. Table 4 and Figure 1 show a negative correlation between 25(OH)D and IL-6 (r = −0.19; P = 0.001), nevertheless the other two inflammatory cytokines (IL-1 β and IFN-α) had no significant correlation with 25(OH)D (P > 0.05). Figure 2 presents in detail the nonlinear relationship between the serum 25(OH)D and IL-6.

Table 3 Serum concentrations of 25(OH)D, IL-6 and odds of TFI-related infertility (n = 289) OR (95% CI), P value

Serum 25(OH)D (ng/mL) ≥20 <20 Serum IL-6 Tertiles (pg/mL) Low (<4.35) Middle (4.35–6.85) High (≥6.85)

Crude

Adjusted*

1 (reference) 2.8 (1.6–4.8), 0.0001†

1 (reference) 4.2 (1.5–11.3), 0.005†

1 (reference) 2.5 (1.3–5.0), 0.008† 8.1 (4.1–15.9), <0.0001†

1 (reference) 3.7 (1.5–9.5), 0.006† 13.2 (4.8–36.4), <0.0001†

25(OH)D, 25-hydroxyvitamin-D; IL, interleukin; TFI, tubal factor infertility. * Adjusted for survey month, age, body mass index, education, family income, sun exposure, parity, vitamin D supplementation in 30 d. † P < 0.05.

There are two plateaus, one of which is <15.6 ng/mL (β = 0.4; 95% CI, −0.2 to 1; P = 0.18), the other is >22 ng/mL (β = 0.1; 95% CI, −0.4 to 0.7; P = 0.68). When 25(OH)D was between these two inflections, the IL-6 concentration significantly decreased with the increase of 25(OH)D concentration (β = −0.7; 95% CI, −1.4 to −0.1; P < 0.001). The interaction analysis reveals that 25(OH)D and IL-6 had an interaction for the risk for TFI after adjusting for confounders. Defining the women with neither high IL-6 (≥P50) nor low 25(OH)D (<20 ng/mL) as the reference, the women with both high IL-6 and low 25(OH)D had the highest risk for TFI (OR, 10.6; 95% CI, 4.2–26.3; P < 0.0001). Meanwhile, women with only high IL-6 (OR, 3.7; 95% CI, 1.6–9.5; P = 0.02) or only low 25(OH)D (OR, 3.3; 95% CI, 1.2–4.6; P = 0.09) had a lower risk for TFI, indicating there might be an interaction between 25(OH)D and IL-6 for TFI (Table 5). Discussion This case–control study revealed that there was an inverse relationship between 25(OH)D and the risk for TFI, and serum 25(OH)D concentration was significantly and negatively correlated with serum IL-6, which also presented a risk factor for TFI. We also found an interaction between IL-6 and 25(OH)D for the risk for TFI-related infertility. Overall, the median concentration of 25(OH)D (18.5 ng/mL) of all women in the current research was well lower than the recommended concentration in adults (≥30 ng/mL), even below the criterion of vitamin D insufficiency (21–29 ng/mL) based on 2011 Endocrine Society guidelines [41,42]. Another study focusing on Chinese women also revealed a similar concentration of 25(OH)D (16.80 ± 5.64 ng/mL) [43]. About 63% of the infertile women in the present study had VDD, which was similar to women described in Belgium (65%) [13] and Germany (59.2%) [44], with mean (±SD) ages of 30.5 (±3.7) and 32.8 (±4.9) y, respectively. Additionally, a lower prevalence of 40.1% was reported in infertile Italian women [21]. The high prevalence of VDD in the women may be due to less likely taking vitamin supplementation and

Table 4 Correlation between serum 25(OH)D concentration and three inflammatory cytokines Variables Serum 25(OH)D (ng/mL)

IL-6 (pg/mL) IFN-α (pg/mL) IL-1 β (pg/mL)

r

95% CI low

95% CI upper

P value

−0.19 −0.04 −0.001

−0.30 −0.15 −0.12

−0.07 0.08 0.11

0.001* 0.52 0.99

25(OH)D, 25-hydroxyvitamin-D; IFN, interferon; IL, interleukin. * P < 0.05.

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Fig. 1. Correlation relationship between serum 25(OH)D concentration and IL-6 concentration (r = −0.19; P = 0.001). 25(OH)D, 25-hydroxyvitamin-D; IL, interleukin.

less sunshine hours [45]. This alarming prevalence of VDD in China is of clinical significance given the recent evidence supporting a critical role of vitamin D in regulating human fertility [23,46]. The median concentrations of three cytokines were 5.3 pg/mL

(4–7.5 pg/mL) for IL-6, 6.7 pg/mL (5.7–7.9 pg/mL) for IFN-α, and 2.8 pg/mL (2.1–3.6 pg/mL) for IL-1 β. Serum IL-6 concentration was slightly higher than the values reported by others (e.g., 3.41 [0–9.12] and 2.39 [1.31–4.26] pg/mL) [47,48]. This discrepancy

Fig. 2. The nonlinear relationship between serum 25(OH)D and IL-6. There are two plateaus, one of which is <15.6 ng/mL (β = 0.4; 95% CI, −0.2 to 1; P = 0.18), the other is >22 ng/mL (β = 0.1; 95% CI; −0.4 to 0.7; P = 0.68). When 25(OH)D was between these two inflections, the IL-6 concentration significantly decreased with the increase of 25(OH)D concentration (β = −0.7; 95% CI, −1.4 to −0.1; P < 0.001). 25(OH)D, 25-hydroxyvitamin-D; IL, interleukin. Adjusted for survey month, age, body mass index, education, family income, sun exposure, parity, vitamin D supplementation in 30 d.

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Table 5 The unadjusted and adjusted interactions of serum 25(OH)D and IL-6 between the control and TFI groups Exposure Serum 25(OH)D (ng/mL)

IL-6 dichotomous (pg/mL)

High (≥20) Low (<20) High (≥20) Low (<20)

Low (
n (%)

Unadjusted, OR (95% CI) P interaction

Adjusted,* OR (95% CI) P interaction

67 (17.9) 81 (28.4) 40 (32.5) 100 (60)

Reference 1.8 (0.8–4) 0.14 2.2 (0.9–5.5) 0.09 6.9 (3.3–14.4) < 0.0001†

Reference 3.3 (1.2–4.6) 0.09 3.7 (1.6–9.5) 0.02† 10.6 (4.2–26.3) < 0.0001†

25(OH)D, 25-hydroxyvitamin-D; IL, interleukin; TFI, tubal factor infertility. * Adjusted for survey month, age, body mass index, education, family income, sun exposure, parity, vitamin D supplementation in 30 d. † P < 0.05.

might be related to the measurement method. However, reports about serum IFN-α and IL-1 β concentrations in Chinese women are still scarce. In the present study, we divided infertility into three groups according to the causes, and analyzed their association with 25(OH)D concentrations. In the three types of female infertility, only TFI women had lower 25(OH)D compared with women who were fertile. TFI is female infertility caused by diseases, obstructions, damage, scarring, congenital malformations, or other factors that impede the descent of a fertilized or unfertilized ovum into the uterus through the fallopian tubes and prevents a normal pregnancy and full-term birth. Tubal factors cause 25% to 35% of female infertility [49,50]. Only one observation provided evidence that TFI was negatively associated with 25(OH)D concentrations, although this result was not statistically significant with a small sample size (n = 66) [21]. Exact mechanisms whereby vitamin D may participate in the regulation of TFI remain far from clear. Investigators have suggested that pelvic inflammatory disease (PID) is the most common cause of tubal disease (50%) [51,52]. Long-term complications of PID are mostly associated with the coinfection of Chlamydia trachomatis and Neisseria gonorrhoeae [53]. The induction of proinflammatory cytokines after infection can damage the epithelium of the fallopian tubes, impede the descent of a fertilized or unfertilized ovum into the uterus through the fallopian tubes, and prevent a normal pregnancy and full-term birth [29,54]. To clarify the changes of proinflammatory cytokines in infertility, we selected three representative factors (IFN-α, IL-6, and IL-1 β) as the objects of measurement according to the literature [29]. IFN-α consists a family of extracellular signaling proteins with demonstrated antiproliferating, immunomodulatory, and antiviral activities [55]. IL-6 and IL-1 β are important mediators of inflammation and mediate many pathways of the normal immune response [56–58]. It has been demonstrated that lower follicular IL-1 β and IFN-α are correlated with TFI [59]; however, we did not find any significant differences in serum IL-1 β and IFN-α concentrations between the fertile and TFI groups. To our knowledge, this is the first time that a significant increase of serum IL-6 concentration in patients with TFI was seen. IL-6 is a pleiotropic cytokine that controls ovarian function and fertility [60]. Refaat et al. reported an increase in the expression of IL-6 and its signaling molecules within human fallopian tubes bearing ectopic pregnancies, and those tubes tissues were concurrently positive for Chlamydia trachomatis [61]. Tubal IL-6 could be involved in the immune response, tubal damage induced by bacterial infections of genital tract, or both. Furthermore, the aberrant increase of IL-6 by the tubal epithelial cells could contribute to the pathogenesis of tubal pregnancy as it has been demonstrated that IL-6 inhibits tubal ciliary beats in vitro [62]. Cytokine polymorphisms were associated with the severity of tubal damage

in women with Chlamydia-associated infertility, genetic predisposition to low IL-6 production might increase the risk for disease complications occurring [63]. Addtionally, some studies also found an association between IL-6 and other infertility types. One study reported on increased concentrations of serum IL-6 in women with unexplained infertility [64]. Pellicer and Albert [65] found that serum and follicular concentrations of IL-6 are elevated in infertile patients with endometriosis. Piltonen et al. discovered that women with PCOS have aberrant production of IL-6 in proliferative and secretory phase endometrium, leading to abnormal signaling in luminal epithelium [66,67]. Furthermore, the polymorphism and the concentration of IL-6 in the seminal plasma are found to be associated with the severity of male infertility [68]. Interestingly, as for women, Bhanoori et al. found no significant association between the IL-6 promoter polymorphism and endometriosis [69]. The interaction analysis indicated that there was a synergistic effect on the risk for TFI when combining 25(OH)D and IL-6 status, however, we were not sure whether vitamin D inhibits the production of IL-6 or vice versa. The existing literature seems to advocate the former idea. As currently understood, vitamin D has antiinflammatory and immunomodulatory activities [30–35,70–72]. Evans et al. reported that 1,25(OH) 2 D 3 and 25(OH)D reduce the concentrations of several decidual proinflammatory cytokines (such as IL-1, IL-6, IFN-γ, TNF, etc.) productions by isolated uterine natural killer cells [73]. Two studies also demonstrated that 1,25(OH)2 D3 inhibits the secretion of IL-6 in monocytes and normal prostate cells [74,75]. Additionally, the study by Tavakoli et al. indicated that IL-6 production by endometrial cells from women with unexplained recurrent spontaneous abortion was significantly reduced by 1,25(OH)2 D3 treatment in vitro [76]. Moreover, currently no literature has been published regarding the effects of IL-6 on the 25(OH)D concentrations, thus the exact causal link is required to be elucidated. The existing literature relating serum vitamin D status with the risk for PCOS is equivocal. Evidence does suggest that VDD might be involved in the pathogenesis of the metabolic syndrome and insulin resistance in PCOS [77,78]. Whether vitamin D also is related to endocrine parameters and fertility in PCOS is less clear. Other observational studies indicate a favorable effect of vitamin D for the reproductive and metabolic health of women with PCOS [79,80]. Similar to our results, some studies also found no significant difference between women with PCOS and controls regarding the concentrations of 25(OH)D [21,81]. Of note, we cannot conclude that there was no benefit of vitamin D in decreasing the incidence or severity of PCOS, as our exploration was limited. Additionally, a complex association also exists between vitamin D and endometriosis. Somigliana et al. reported that endometriosis is associated with higher serum concentrations of

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25(OH)D [82], whereas in a large prospective study, a significantly lower rate of endometriosis was suggested among women with higher plasma 25(OH)D concentrations [83]. Our result supports the latter suggestion, although our inverse association was not statistically significant. We speculate that the negative result may be due to the small sample size of the endometriosis group. Because endometriosis is an autoimmune disease, we hypothesized that vitamin D as an immunomodulatory might decrease the prevalence of this condition [84]. However, the exact nature of the relationship between endometriosis and the 25(OH)D remains to be clarified. This case–control study evaluated the potential relationship among 25(OH)D status, inflammatory cytokines, and the risk for infertility. The limitations are worth noting. First, a case– control study design provides a challenge in terms of determining whether the associations among 25(OH)D, IL-6, and TFI are causal. Second, the infection rate of Chlamydia trachomatis and other potential risk factors were not fully investigated. Third, we did not administer a food-recall questionnaire. Finally, serum 25(OH)D was measured only at the time of diagnosis, which does not necessarily reflect vitamin D levels during the entire period in which infertility occurs. These should be considered and included in future studies. Conclusions The results of the present study demonstrated that both VDD and high serum IL-6 concentration are risk factors for TFI in Chinese women, whereas serum 25(OH)D concentration was significantly and negatively correlated with serum IL-6. Furthermore, there was an interaction between IL-6 and 25(OH)D for the risk for TFI-related infertility. Combined current results with previous literature, we hypothesized that vitamin D might reduce the risk for TFI through suppressing the production of IL-6. Prospective studies and clinical trials are warranted to assess the causal nature of these relationships. Acknowledgments The authors acknowledge Fangfang Guo and Liangnian Song for a critical reading of the manuscript and useful comments. References [1] Mascarenhas MN, Flaxman SR, Boerma T, Vanderpoel S, Stevens GA. National, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys. PLoS Med 2012;9:e1001356. [2] Thonneau P, Marchand S, Tallec A, Ferial ML, Ducot B, Lansac J, et al. Incidence and main causes of infertility in a resident population (1,850,000) of three French regions (1988–1989). Hum Reprod 1991;6:811–16. [3] Templeton A. Infertility and the establishment of pregnancy—overview. Br Med Bull 2000;56:577–87. [4] Cui W. Mother or nothing: the agony of infertility. Bull World Health Organ 2010;88:881–2. [5] Chachamovich JR, Chachamovich E, Ezer H, Fleck MP, Knauth D, Passos EP. Investigating quality of life and health-related quality of life in infertility: a systematic review. J Psychosom Obstet Gynaecol 2010;31:101–10. [6] Poppe K, Velkeniers B. Female infertility and the thyroid. Best Pract Res Clin Endocrinol Metab 2004;18:153–65. [7] Panda DK, Miao D, Tremblay ML, Sirois J, Farookhi R, Hendy GN, et al. Targeted ablation of the 25-hydroxyvitamin D 1 alpha -hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci USA 2001;98:7498–503. [8] Johnson LE, DeLuca HF. Vitamin D receptor null mutant mice fed high levels of calcium are fertile. J Nutr 2001;131:1787–91. [9] Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, Seino Y, et al. Is an important factor in estrogen biosynthesis of both female and male gonads 1. Endocrinology 2000;141:1317–24.

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