Relationship Between Vitamin D and Hormones Important for Human Fertility in Reproductive-Aged Women

Vitamin D deficiency is very common in women of reproductive age. Studies in animals suggests a link between vitamin D and reproductive hormone biosynthesis. A systematic analysis of the correlation of reproductive hormones in reproductive-aged women with both total and free vitamin D was, however, not done so far. This cross-sectional study was performed in 351 healthy reproductive age Caucasian women (median age, 28.0 years; interquartile ranges, 24.7-31.0 years). We measured serum levels of both total and free 25(OH)D, endocrinological, hematological and biochemical parameters. Spearman’s rank correlations were performed to assess the correlation between 25(OH)D metabolites and selected parameters. Total vitamin D and free vitamin D measurements correlated well (rho=0.912, p < 0.0001). Both total 25(OH)D and free 25(OH)D showed significant negative correlation with FAI (rho=-0.229, p<0.0001 and rho=-0.195, p<0.0001 for total and free 25(OH)D, respectively); LH (rho=-0.177, p=0.001 and rho=-0.114, p=0.04 for total and free 25(OH)D, respectively), testosterone (rho=-0.174, p=0.001 and rho=-0.190, p<0.0001 for total and free 25(OH)D, respectively) and AMH (rho=-0.130, p=0.015 and rho=-0.107, p=0.047 for total and free 25(OH)D, respectively). Our study showed comparable correlations of both total and free 25(OH)D with endocrinological parameters, i.e. inverse correlations with free androgen index, luteinizing hormone, testosterone, LH/FSH ratio, androstenedione and anti-Müllerian hormone, and also with hematological and biochemical parameters, i.e. inverse correlations with erythrocytes, hsCRP and leukocytes count, and positive correlation with transferrin saturation, mean corpuscular hemoglobin and mean corpuscular volume in healthy reproductive age women.

Vitamin D deficiency is very common in women of reproductive age. Studies in animals suggests a link between vitamin D and reproductive hormone biosynthesis. A systematic analysis of the correlation of reproductive hormones in reproductive-aged women with both total and free vitamin D was, however, not done so far. This cross-sectional study was performed in 351 healthy reproductive age Caucasian women (median age, 28.0 years; interquartile ranges, 24.7-31.0 years). We measured serum levels of both total and free 25(OH)D, endocrinological, hematological and biochemical parameters. Spearman's rank correlations were performed to assess the correlation between 25(OH)D metabolites and selected parameters. Total vitamin D and free vitamin D measurements correlated well (rho=0.912, p < 0.0001). Both total 25(OH)D and free 25(OH)D showed significant negative correlation with FAI (rho=-0.229, p<0.0001 and rho=-0.195, p<0.0001 for total and free 25(OH)D, respectively); LH (rho=-0.177, p=0.001 and rho=-0.114, p=0.04 for total and free 25(OH)D, respectively), testosterone (rho=-0.174, p=0.001 and rho=-0.190, p<0.0001 for total and free 25(OH)D, respectively) and AMH (rho=-0.130, p=0.015 and rho=-0.107, p=0.047 for total and free 25(OH)D, respectively). Our study showed comparable correlations of both total and free 25(OH)D with endocrinological parameters, i.e. inverse correlations with free androgen index, luteinizing hormone, testosterone, LH/FSH ratio, androstenedione and anti-Müllerian hormone, and also with hematological and biochemical parameters, i.e. inverse correlations with erythrocytes, hsCRP and leukocytes count, and positive correlation with transferrin saturation, mean corpuscular hemoglobin and mean corpuscular volume in healthy reproductive age women.
Keywords: total 25-hydroxyvitamin D, measured free 25-hydroxyvitamin D, fertility, hormones, reproductive-aged women INTRODUCTION Vitamin D deficiency is very common in women of reproductive age (1,2). The physiological role of vitamin D in reproduction remains ambiguous. Early animal experiments found that 25 (OH)D deficiency rats showed a compromised mating behavior, reduced fertility rates, decreased litter sizes and impaired neonatal growth, suggested the importance of vitamin D in reproduction (3)(4)(5). In human, low maternal vitamin D status has been associated with adverse maternal and fetal outcomes, including preeclampsia (6,7), gestational diabetes mellitus (8,9), small for gestational age (10) low birth weight (11).
Vitamin D appears to function through a single vitamin D receptor (VDR), which also has been identified in human female several reproductive tissues (12). Several studies reported that vitamin D deficiency affects both insulin secretion and insulin resistance. Insulin resistance is a hallmark of the polycystic ovarian syndrome, one of the most common endocrine disorders that affects reproductive age women (13)(14)(15)(16).
As of today, measurements of total 25(OH)D are the most widely used way to assess vitamin D status in daily clinical practice. Serum 25(OH)D consists of three main metabolites, about 85 to 90% are bound to its specific carrier-vitamin D bound protein (DBP), about 10 to 15% are bound to serum albumin, only less than 0.1% exists as fully free unbound form (17,18). However, only free vitamin D is bioactive, because it can pass freely the lipophilic cell membrane and interacts with the nuclear vitamin D receptor. Total vitamin D concentrations depends DBP concentrations, which depends itself on liver function, kidney function, endocrine status and even ethnicity rather than mineral-bone metabolism (19)(20)(21)(22). Thus, total vitamin D concentrations may not entirely reflect the vitamin D status due to its above listed confounding factors. Free 25 (OH)D might be a more reliable marker of vitamin D status in the body (23). This hypothesis is supported by recent studies (18,24).
The associations of total 25(OH)D with sex steroid hormones were inconsistently reported in either women with PCOS or healthy women (25)(26)(27)(28). We speculate that disparities in DBP levels in previous studies may led to the inconsistent associations between vitamin D and sex hormones. To the best of our knowledge, no information is available in human on the correlations of both total 25(OH)D and directly measured free 25(OH)D with reproduction related endocrinological parameters. We thus carried out a cross sectional study to compare total 25(OH)D and directly measured free 25(OH)D in a relatively large cohort of healthy reproductive age women (n=351). We evaluated correlations of 25(OH)D metabolites with reproduction related endocrinological parameters including follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid hormones, anti-Müller hormone (AMH), sex steroid hormones and also hematological and biochemical parameters.

Study Participants
The study was a part of the Berlin Birth Cohort (BBC) study (29)(30)(31). This study was approved by the local ethical committee of the Charité-Universitätsmedizin Berlin, Germany. The study cohort consisted of 351 healthy Caucasian women of reproductive age asking for endocrine evaluation getting pregnant in four endocrine outpatients' facilities in Berlin, Germany. Blood was taken between day 3 and 6 of the menstrual cycle. Patients with hypertension, any sign of heart/liver/kidney diseases, diabetes type 1 or 2 or any chronic disease such as rheumatoid arthritis and obesity were not asked to participate. All patients gave their written informed consent.
All biochemical and endocrine parameters measurements were analyzed in a certified clinical laboratory, IFLb -Institute für Laboratoriumsmedizin Berlin (https://www.iflb.de/das-labor/), which has committed to quality management in accordance with the requirements of the DIN EN 15189 standard, verified by regular system and process reviews and monitoring by the German Accreditation Service (DAkkS) (accreditation number D-ML-13224-01). All measurements are subject to daily quality controls. External standards provided by an organization of the German clinical chemistry association are included in the daily measurements. All methods were performed in accordance with relevant guidelines and regulations.

Statistical Analysis
Each parameter was examined for normality using the D'Agostino test, and most parameters were found to be not normally distributed (p <0.05). Descriptive statistics are presented as medians (interquartile ranges). Spearman's rank correlation coefficients were performed to assess the correlation between 25(OH)D metabolites and selected hematological, biochemical and endocrinological parameters. Correlations were regarded significant if p-values were lower than 0.05. All analysis was performed using SPSS version 25.0 (Chicago, IL, USA) and GraphPad Prism version 8.0 (San Diego, CA, USA).

Associations Between Free 25(OH)D and Total 25(OH)D
The positive and strong correlation between total 25(OH)D and free 25(OH)D was statistically significant (rho=0.912, p < 0.0001, Figure 1).

Correlations Between Total 25(OH)D and Free 25(OH)D With Endocrinological Parameters
Spearman's correlations between endocrinological parameters and 25(OH)D metabolites are presented in Table 2. Both total 25(OH)

DISCUSSION
This cross-sectional study compared total 25(OH)D and directly measured free 25(OH)D in healthy reproductive age women.  Total vitamin D and free vitamin D measurements correlated well. Both showed comparable correlations with reproduction related endocrinological, hematological parameters, liver function parameters, thrombophilia parameters and anemia related parameters (see Tables 2 and 3).
Increasing evidences show that vitamin D may play an important role in regulating female fertility (33)(34)(35)(36). Low vitamin D status is associated with adverse maternal and fetal outcomes (6)(7)(8)(9)(10)(11), and is involved in the development of specific gynecological conditions that affecting fertility, such as endometriosis and polycystic ovarian syndrome (PCOS) (14,37,38). Several animal and human studies have linked vitamin D metabolism with sex steroid synthesis (25,39). In the present study we observed that both total and free 25(OH)D were inversely correlated with FAI, LH, total testosterone, AMH, androstenedione, TSH, and positively correlated with SHBG in reproductive-age women.
It is a key finding of the current study that laboratory parameters describing androgens such as free androgen index (FAI), total testosterone and androstenedione are among the parameters with the strongest negative correlation between either free or total vitamin D in reproductive-age Caucasian women. Testosterone has been extensively studied in male, and considerable evidence supports the influence of vitamin D on semen quality through the regulation of calcium metabolism and testosterone production (39). However, few studies have focused on the effects of vitamin D on testosterone in women. Our results showed that both total and free 25(OH)D were negatively correlated with testosterone. Our data are in agreement with another study showing a negative correlation between total 25 (OH)D and free T as well as total T in the follicular fluid of healthy women (40) and with a meta-analysis of studies in women with PCOS (41) showing likewise an inverse relationship of testosterone to total vitamin D. There is, however, a small study in healthy women showing a positive correlation between androgens and total vitamin D (25). Given the size of this study eight times smaller than our study and the other studies in PCOS women and the study analyzing ovarian fluid, see above, it is more likely that this small study was simply underpowered.
From a biochemical aspect, both androgens and vitamin D belong to the steroid family. As mentioned above, we report a negative correlation between laboratory parameters describing androgens and vitamin D. A potential explanation of such a correlation could be derived from the ability of vitamin D to act as a regulator of a number of enzymes involved in the regulation of the production of adrenal steroid hormones including adrenal androgens as well as ovarian sex hormones (42). Sex hormones are produced in the gonads either by in situ synthesis from cholesterol or by enzyme catalyzed conversion of androstenedione or DHEA which are excreted to the circulation from the adrenal gland. Androgens (e.g. testosterone) are produced via reactions catalyzed by 17b-hydroxysteroid dehydrogenase (17b-HSD). The expression of this enzyme was reported to be regulated by 1a,25dihydroxyvitamin D 3 in human prostate cell lines and keratinocytes (43,44). Moreover, 1a,25-dihydroxyvitamin D 3 was reported to exert tissue-specific effects on androgen metabolism where it led to increased androgen production in breast cancer cells, however, dihydrotestosterone production was decreased in adrenocortical cells treated with 1a,25-dihydroxyvitamin D 3 (45).
Our observational clinical data might suggest that treatment with vitamin D might be a way to improve the endocrine status in women with hyperandrogenism. However, two meta-analyses have reported controversial findings with respect to the effect of vitamin D supplementation on androgens in women with PCOS (46,47). This could be due to two circumstances. First, the vitamin D status in these studies was determined using total vitamin D. Second, the optimal doses to improve the androgen status in humans are still unknown. Proper dose finding studies would be needed. However, this was not done. Vitamin D concentrations for an optimal androgen status, fertility rate and embryo development in humans are currently unknown. PCOS -beside high androgens -is also characterized by elevated LH and AMH (48,49). Both are also inversely correlated to free and total vitamin D. This again underlines the possible importance of vitamin D to optimize the endocrine situation in women with PCOS. As mentioned above, this would require adequately designed dose-finding studies. The present study also indicated an association between 25 (OH)D and anemia related parameters, which is in line with some previous studies (50)(51)(52)(53)(54). And we also found that both vitamin D metabolites are negatively correlated with hsCRP, which draw attention to the hypothesis that low 25(OH)D maybe involved in the chronic inflammation. Low grade inflammation is an unfavorable factor when aiming to become pregnant and also harmful for the developing fetus (55)(56)(57).
The strength of correlation of free and total 25(OH)D and endocrinological, hematological and biochemical parameters was similar in healthy reproductive age women. Thus, both ways of analyzing the vitamin D status in healthy reproductive age women with regard to the evaluation of endocrine fertility parameters are suitable tools. The effect of estrogens on vitamin D binding protein and hence the relationship of free and total 25(OH)D is obviously negligible in a study population of healthy women with the same genetic background. This might be different in populations with mixed ethnic background and underlying diseases with known effects on serum vitamin D binding protein concentrations such as kidney diseases or liver diseases (5,17). Kidney diseases and major liver diseases are usually rare in young women, thus the use of both ways of analysing the vitamin D status in this particular population is justified. If these young women get pregnant, the situation changes, then, the rising levels of female steroids hormones do substantially induce hepatic synthesis of vitamin D binding protein (58) and hence free 25(OH)D is much better correlated with markers of bone metabolism (calcium, BSAP), lipid metabolism (adiponectin, LDL cholesterol, LDL/HDL ratio) and kidney function (urea) than total 25(OH)D (32).
A strength of our study represents its large sample size of Caucasian women. Furthermore, we measured both total and free 25(OH)D. We acknowledge that a cross-sectional study design limits drawing conclusion on causality, and the lack of clinical data such as previous obstetric/gynecological history, body mass index and blood pressure represents a study limitation.
In conclusion, this is the first study to compare total 25(OH)D and directly measured free 25(OH)D in a relatively large cohort of healthy reproductive age women. Total and free vitamin D showed similar correlations with reproduction related endocrinological parameters, hematological parameters, liver function parameters, thrombophilia parameters and anemia related parameters in healthy reproductive age women. In particular androgens but also LH and AMH are inversely correlated with both free and total 25 (OH)D.
In conclusion, this is the first study to compare total 25(OH)D and directly measured free 25(OH)D in a relatively large cohort of healthy reproductive age women. Total and free vitamin D showed similar correlations with endocrinological parameters, i.e. inverse correlations with free androgen index, luteinizing hormone, testosterone, LH/FSH ratio, androstenedione and anti-Müllerian hormone, and also with hematological and biochemical parameters, i.e. inverse correlations with erythrocytes, hsCRP and leukocytes count, and positive correlation with transferrin saturation, mean corpuscular hemoglobin and mean corpuscular volume in healthy reproductive age women.

DATA AVAILABILITY STATEMENT
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ETHICS STATEMENT
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