Vitamin B12 Is Associated with Higher Serum Testosterone Concentrations and Improved Androgenic Profiles Among Men with Infertility

Background Infertility impacts 16% of North American couples, with male factor infertility contributing to ∼30% of cases. Reproductive hormones, especially testosterone, are essential for spermatogenesis. An age-independent population-level decline in testosterone concentrations over the past few decades has been proposed to be a consequence of diet and lifestyle changes. Vitamin B12 is present in the testes and has been suggested as an adjuvant nutritional therapy for male infertility due to its potential to improve sperm parameters. However, evidence examining the relationship between vitamin B12 and reproductive hormones is limited. Objectives The objective was to cross-sectionally examine the relationship between serum vitamin B12 and male reproductive hormones (luteinizing hormone, follicular stimulating hormone, total testosterone, estradiol, and prolactin). Methods Men with infertility (n = 303) were recruited from Mount Sinai Hospital in Toronto, Canada. Serum was analyzed for vitamin B12 and reproductive hormones. Statistical analyses included nonparametric Spearman’s rank correlation coefficient, linear regression, logistic regression, and effect modification by age and BMI linear regressions. Results An independent monotonic relationship between serum vitamin B12 and total testosterone (ρ = 0.19, P = 0.001) was observed. Serum vitamin B12 was linearly associated with total testosterone (unadjusted β = 0.0007, P = 0.008 and adjusted β = 0.0005, P = 0.03). Compared to individuals in the lowest tertile of serum vitamin B12, those in the middle tertile (adjusted odds ratio [OR] = 0.48; 95% confidence interval [CI]: 0.25, 0.93, P = 0.03) and the highest tertile (unadjusted OR = 0.41; 95% CI: 0.22, 0.77, P = 0.005 and adjusted OR = 0.44; 95% CI: 0.22, 0.87, P = 0.02) had reduced odds of testosterone deficiency. Conclusions These findings suggest that among men with infertility, low serum vitamin B12 is associated with a higher risk of testosterone deficiency and impaired androgenic hormonal profiles that impact spermatogenesis and consequently, fertility.


Introduction
Infertility is characterized as the inability to achieve clinical pregnancy after a year of consistent unprotected sexual intercourse between a man and woman assigned to those genders at birth.Approximately 16% of couples in North America are affected by infertility, with male factor infertility accounting for ~30% of cases and a combination of male and female factors contributing to another 20% of cases [1].The decline in male fertility, characterized by factors such as diminished sperm quality and hypogonadism, has become a growing concern [2,3].
Previous research has identified a significant decrease in testosterone concentrations among males, independent of age, attributed to various influences including health, environmental factors, and dietary choices [2,4].Although endocrine dysfunctions, physical impairments, and genetic polymorphisms were traditionally considered primary causes of male infertility, recent studies have shed light on the role of lifestyle factors such as sleep disruptions, body weight, smoking, exposure to environmental toxins, and nutrition [5][6][7][8][9][10][11][12][13][14][15].Promising evidence suggests that adopting a dietary pattern, characterized by a higher consumption of fruits and vegetables while limiting meat, fat, refined sugars, and processed foods, can positively impact sperm parameters [16][17][18][19].Vitamin B 12 has gained attention for its potential impact on male reproductive function, as highlighted in a recent review investigating the influence of nutrition and genetics on male fertility [20].
Seminal parameters and reproductive hormones are commonly used as indicators of male fecundity and for diagnosing male factor infertility [21,22].Although existing research in the field of nutrition and male fertility primarily focuses on seminal parameters, there is a significant knowledge gap regarding the influence of micronutrients on male reproductive hormones, which play a crucial role in spermatogenesis and overall male reproductive health.The hypothalamic-pituitary-gonadal (HPG) axis serves as the principal signaling pathway responsible for regulating reproductive hormones, thus overseeing spermatogenesis [23].Within this HPG axis, the hypothalamus releases gonadotropin-releasing hormone, which stimulates the secretion of gonadotropins, follicular stimulating hormone (FSH), and luteinizing hormone (LH) [23].FSH primarily acts on Sertoli cells in the testes, supporting spermatogenesis and the maturation of sperm cells, whereas LH stimulates Leydig cells to produce intratesticular androgens, including testosterone, which is essential for spermatogenesis [23].Androgens can also be converted into estrogens, particularly estradiol, in the testes and peripheral tissues through the action of aromatase (CYP19) [24].Elevated concentrations of estradiol can potentially hinder fertility [25].Another hormone pertinent to male reproductive health is prolactin, which inhibits the HPG axis, leading to reduced testosterone synthesis [26].
Vitamin B 12 (cobalamin), is an essential water-soluble vitamin critical to several physiological functions such as DNA synthesis and red blood cell maturation.Some studies have suggested vitamin B 12 as adjuvant nutritional therapy for male infertility due to its potential to improve sperm health [20,27].Methylcobalamin, the active form of vitamin B 12 , plays a critical role in homocysteine's remethylation cycle [28].It acts as an essential cofactor for methionine synthase, facilitating the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine in the conversion pathway from homocysteine to methionine [28].Hyperhomocystenenmia, often attributable to low vitamin B 12 and folate concentrations, has been associated with oxidative stress and diminished fertility parameters including increased DNA fragmentation and reduced successful in vitro fertilization, reduced sperm concentration, and diminished sperm motility [18,[29][30][31].A review by Banihani et al. [27] on vitamin B 12 summarized some limited existing research, which demonstrated a positive association between vitamin B 12 and improved sperm count, sperm motility, and DNA integrity.Such findings can be attributable to the influence of vitamin B 12 on homocysteine, enhanced control of nuclear factor-κB, decreased spermatozoa energy production, improved reproductive organ functionality, decreased nitric oxide production, and reduced inflammation-induced sperm impairments [27].However, there is contrary evidence from other observational studies and clinical trials that have found no association between vitamin B 12 and semen quality [32][33][34].For example, a cross-sectional study by Jathar et al. [34] found that although mean seminal plasma vitamin B 12 concentrations were lower in individuals belonging to the azoospermic group compared with individuals belonging to the normospermic and oligospermic group, seminal plasma vitamin B 12 concentrations were not associated with sperm parameters, including, sperm count, motility, or morphology.Only one study involving a small group of 26 males with infertility has examined the relationship between vitamin B 12 and reproductive hormones [35].A comprehensive review on vitamin B 12 and male reproductive health, highlighted this scarcity in research on the impact of vitamin B 12 on reproductive hormones [27].Therefore, the objective of the present study was to assess the association between serum vitamin B 12 concentrations and reproductive hormones in a population of men experiencing infertility.

Study design and participants
This study employed a cross-sectional design to assess the relationship between serum vitamin B 12 concentrations and reproductive hormones.A cohort of 832 males experiencing infertility was recruited with informed consent, from the Murray Koffler Urologic Wellness Center at Mount Sinai Hospital in Toronto, Canada.The recruitment phase from June 2019 to August 2021, utilized a clinic-based recruitment strategy where participants were enrolled as they attended their clinic appointments.Eligibility criteria excluded individuals who had undergone postoperative procedures; experienced physical impairments leading to infertility; were unable to provide venous blood or semen samples; had missing data; had recently used fertility-related medication within the last 6 mo, had undergone vasectomy; received testicular cancer radiation therapy <4 y ago; or had conditions such as Klinefelter Syndrome, Y chromosome microdeletions, or cystic fibrosis.After applying these exclusions, the final sample size consisted of 303 males (Supplemental Figure 1).Bloodwork and hormonal assays were missing at random due to challenges with accessing laboratory services during the COVID-19 pandemic.The present study adhered to ethical standards set by the University of Toronto Research Ethics Board and Mount Sinai Hospital Research Ethics Board.Approval was obtained from these relevant committees overseeing research involving human participants.All participants provided written informed consent to participate in the study.

Anthropometric measurements and general health information
Participants completed a computerized personal health questionnaire provided by Mount Sinai Hospital's Men's Health Institute.The questionnaire gathered information on anthropometric measurements, demographic details, medical history, lifestyle data, and female partner medical history.Further information regarding the specific components and details of the questionnaire have been previously described [36].

Nutritional and reproductive hormone biochemical measurements
Serum vitamin B 12 and reproductive hormone concentrations were assessed by collecting venous blood samples from the participants.The analyses were conducted at either the onsite laboratory of Mount Sinai Hospital or at LifeLabs medical laboratory services.The reproductive hormones included in the analyses were FSH, LH, total testosterone (TT), prolactin, and estradiol.These measurements were conducted using ELISA assays in accordance with the laboratory's standard operating procedures.Estradiol concentrations below 93 pmol/L were reported as values < 93 pmol/L by the laboratory.

Statistical analyses
The statistical software R Studio (Version 1.1.463)was utilized for the data analyses.Statistical significance was defined as P < 0.05.Descriptive statistics, including counts and the corresponding percentages relative to the total sample as well as mean (AESD), were used to summarize subject characteristics with categorical and continuous variables.Differences between the tertiles of serum vitamin B 12 groups were assessed using chisquare test and analysis of variance for categorical variables and continuous variables, respectively.
The monotonic relationship between serum vitamin B 12 concentrations and reproductive hormones, excluding estradiol, was evaluated using nonparametric Spearman's rank correlation coefficient analyses.The Shapiro-Wilk test was employed to examine normality.For parametric analyses, square root transformation was applied to reproductive hormones to normalize skewed distributions.Residual distributions of all models were assessed for normality and variance inflation factor was computed to assess for collinearity.For initial parametric analyses, linear regression models were used to assess the association between serum vitamin B 12 concentrations and reproductive hormones as continuous variables.Both unadjusted and covariate-adjusted linear regression analyses were completed.To explore potential effect modification, additional linear regression models were constructed by incorporating interaction terms for age and serum vitamin B 12 concentrations, as well as for BMI and serum vitamin B 12 concentrations.For any model(s) showing significant interactions, simple slope and false discovery rate-corrected Johnson-Neyman interval analyses were conducted.
Further analyses were completed where logistic regression models were employed, categorizing serum vitamin B 12 concentration into tertiles and dichotomizing reproductive hormones into clinically significant categories.Serum vitamin B 12 concentration was grouped into tertiles instead of being categorized based on deficiency and elevated concentration cutoffs.This approach was selected because deficiency and elevated clinical cutoffs for serum vitamin B 12 are established based on diseaserelated epidemiological data and markers unrelated to fertility.The clinical cutoffs for reproductive hormones were determined based on values outside their respective normal ranges: elevated FSH (>12.4IU/L), elevated LH (>7.8 IU/L), low TT (<9.2 nmol/ L), elevated prolactin (>18 ng/mL), and elevated estradiol (>146.8pmol/L).In the multivariate linear and logistic regression models, the clinically relevant covariates adjusted for were age (y), BMI (kg/m 2 ), current alcohol consumption (yes/no/unspecified), current smoking status (yes/no), meteorological season blood was drawn (categorized as winter, spring, summer, and fall), and ethnicity (Caucasian, African-Canadian, Asian, Hispanic, Indo-Canadian, Middle-Eastern, and unspecified).
Table 2 presents the results of the nonparametric Spearman's rank correlation coefficient analyses, revealing a statistically significant and independent monotonic positive association between serum vitamin B 12 concentrations and TT (ρ ¼ 0.19, P ¼ 0.001).Spearman's rank correlation coefficient analyses indicate that higher vitamin B 12 concentrations were associated with higher TT concentrations.No associations were observed between vitamin B 12 concentrations and all remaining reproductive hormones (LH, FSH, and prolactin).
Assessment of the linear relationship between serum vitamin B 12 concentrations and square root transformed reproductive hormones using linear regressions is presented in Table 3.Initially, in the unadjusted univariate analyses a positive association between serum vitamin B 12 concentrations and TT (β ¼ 0.0007, P ¼ 0.008) was observed.In the adjusted multivariate model, the positive relationship between serum vitamin B 12 concentrations and TT remained significant (β ¼ 0.0005, P ¼ 0.03).No other statistically significant associations were observed between serum vitamin B 12 concentrations and reproductive hormones (LH, FSH, prolactin, and estradiol) in both univariate and multivariate linear regression analyses.In covariate-adjusted linear regression analyses, the potential effect modification of age and BMI was explored.In the regression analyses with inclusion of age as an effect modifier, there was no significant interaction between age and vitamin B 12 concentrations.In similar analyses, but with the inclusion of BMI as an effect modifier, there was also no significant interaction between BMI and serum vitamin B 12 concentrations.Since there were no significant interactions for effect modification, simple slope and false discovery rate-corrected Johnson-Neyman interval analyses were not conducted.Thus, both age and BMI did not modify the linear relationship between serum vitamin B 12 concentrations and reproductive hormones.Results for effect modification analyses are presented in Table 3. 2 Differences between groups were compared using chi-square for categorical variables and analysis of variance for continuous variables. 3Serum vitamin B 12 concentration cutoffs are <148 pmol/L, !148 to <701 pmol/L, and !701 pmol/L for deficient, optimal, and elevated, respectively. 4Seasonal variation was determined based on meteorological seasons blood was drawn for analysis, "Winter" ¼ December 1 to February 28/29; "Spring" ¼ March 1 to May 31; "Summer" ¼ June 1 to August 31; "Fall" ¼ September 1 to November 30. 5 Clinical status cutoffs were based on reproductive hormones not within normal range: elevated FSH (>12.4IU/L), elevated LH (>7.8 IU/L), low TT (<9.2 nmol/L), elevated prolactin (>18 ng/mL), and elevated estradiol (>146.8pmol/L).
Results from the logistic regression analyses assessing the association between tertiles of vitamin B 12 concentrations (with the lowest tertile as the reference group) and the clinical status of reproductive hormones (with normal hormone concentrations as the reference group) are presented in Table 4 and Supplemental Figure 2.For LH, in the unadjusted analyses, individuals in the highest tertile of serum vitamin B 12 concentrations had reduced odds of elevated LH compared with those in the lowest tertile of serum vitamin B 12 concentrations (odds ratio [OR] ¼ 0.50; 95% confidence interval [CI]: 0.28, 0.90, P ¼ 0.02).However, the association was no longer significant in the adjusted model (OR ¼ 0.53; 95% CI: 0.25, 1.13, P ¼ 0.05).Compared with individuals in the lowest tertile of serum vitamin B 12 concentration, there was a lower odds of TT deficiency among those in the mid-tertile (OR ¼ 0.48; 95% CI: 0.25, 0.93, P ¼ 0.03) and upper tertile (OR ¼ 0.44; 95% CI: 0.22, 0.87, P ¼ 0.02) of serum vitamin B 12 concentration.For all other remaining reproductive hormones, in both unadjusted and adjusted analyses, there were no significant differences in the odds of experiencing hormone concentrations outside the normal range based on the tertiles of serum vitamin B 12 concentrations.Prolactin was not included in the logistic regression analyses due to minimal variability (<10%) in elevated prolactin concentrations among the study participants.Abbreviations: BMI, body mass index; FSH, follicular stimulating hormone; LH, luteinizing hormone; SE, standard error; TT, total testosterone. 1Reproductive hormones (FSH, LH, TT, and prolactin) are square root transformed.The relationship depicted is the association between serum ascorbic acid concentration and square root of reproductive hormones (FSH, LH, TT, and prolactin). 2 Model adjusted for covariates: age, alcohol consumption, BMI, ethnicity, seasonal variation, and smoking status. 3 Interaction P for the adjusted linear regression model assessing the association between serum vitamin B 12 concentration and serum reproductive hormones with effect modification by age (age by vitamin B 12 interaction term). 4Interaction P for the adjusted linear regression model assessing the association between serum vitamin B 12 concentration and serum reproductive hormones with effect modification by BMI (BMI by vitamin B 12 interaction term). 5Estradiol was categorized into groups: normal ( 146.8 pmol/L) and elevated (>146.8pmol/L).

TABLE 4
Odds ratios (ORs) and 95% confidence intervals (CIs) for the association between tertiles of serum vitamin B 12 concentration and reproductive hormones outside of normal range 1 using binomial logistic regressions.

Discussion
A few studies have examined the association between serum vitamin B 12 concentrations and male reproductive hormones.Our findings show a positive linear association between vitamin B 12 and serum TT concentrations.In a recent study investigating the effects of ascorbic acid on reproductive hormones, an agedependent effect of ascorbic acid on TT was found [36].However, in the present study, we did not observe any modifications in the linear relationship between serum vitamin B 12 and TT based on age or BMI.Additionally, we discovered that individuals in either the mid-or highest tertiles of serum vitamin B 12 had significantly lower odds of experiencing testosterone deficiency compared with those in the lowest tertile of vitamin B 12 .Although the highest tertile showed the lowest odds, it is noteworthy that even individuals in the mid-tertile experienced a significant reduction in the odds of TT deficiency.This suggests that even a slight increase in vitamin B 12 concentrations may contribute to a notable decrease in the likelihood of testosterone deficiency.Although vitamin B 12 showed a significant positive linear relationship with TT concentrations, there was no evidence of a similar association with the other reproductive hormones (FSH, LH, prolactin, and estradiol).Furthermore, there was no significant association between tertiles of serum vitamin B 12 and the odds of experiencing elevated concentrations of FSH, LH, and estradiol.These results suggest that the influence of vitamin B 12 on hormonal regulation may be specific to testosterone.
To our knowledge, only one previous study examined the association between vitamin B 12 administration and male reproductive hormones [35].Isoyama et al. [35] conducted a clinical trial with a group of 26 infertile males and found daily administration of 1500 μg of methylcobalamin for 4-24 wk resulted in some improvements in sperm parameters but no changes in serum FSH, LH, or testosterone.However, that study had several limitations, including the absence of a control group, a limited sample size (n ¼ 26), a variable intervention duration (4-24 wk), no measurement or reporting of serum vitamin B 12 concentration, lack of measurement of compliance to B 12 supplementation, and no consideration for dietary vitamin B 12 intake [35].In addition, there was no assessment of baseline vitamin B 12 concentrations.Without information on baseline vitamin B 12 status, supplementation may not have any discernible impact, especially if individuals already had elevated B 12 concentrations at baseline [35].
A previous study compared the effects of vitamin B 12 and methotrexate administration, alone or in combination, on hormone concentrations in rodents [37].In comparison with the control group, testosterone concentrations increased, but FSH and LH concentrations remained unchanged after administration of vitamin B 12 [37].This is similar to the present study's findings where a linear relationship between serum vitamin B 12 and testosterone was found, but no significant relationship between serum vitamin B 12 and either LH or FSH was found.Their findings suggest that vitamin B 12 may have a protective effect by reducing apoptosis and alleviating endoplasmic reticulum stress associated with testicular toxicity as vitamin B 12 supplementation resulted in improved hormone concentrations, and supported the production of normal germ cells even in the presence of normal hormone concentrations [37].
Despite previous research suggesting men with infertility may have lower serum vitamin B 12 concentrations compared to men who do not experience infertility, the prevalence of vitamin B 12 deficiency was low in the current study population at 0.7% [38,39].This is lower than the 1.2% prevalence of vitamin B 12 deficiency reported in the Canadian general population by the Canadian Health Measures Survey [40].However, a substantial number of the study participants, specifically 12.2%, exhibited elevated serum vitamin B 12 concentrations.
The present study has several strengths.It is the first comprehensive investigation exploring the relationship between serum vitamin B 12 and several key male reproductive hormones in humans.Furthermore, clinically significant covariates were predefined, rather than employing a data-driven approach.Additionally, these covariates were accounted for in statistical analyses, and the utilization of a nutrient biomarker minimized potential self-reporting biases associated with dietary intake assessments.However, certain limitations should be acknowledged.Given the cross-sectional nature of the study design, establishing causality and temporality is not possible.Furthermore, all demographic and anthropometric data relied on selfreporting, introducing a potential risk of self-reporting bias for some of the covariates.In addition, the present study was designed to examine the independent association between vitamin B 12 and reproductive hormones.However, the combined influence of several micronutrients and macronutrients could potentially collectively influence reproductive hormones differently.Although several critical factors were adjusted for in the analyses, unidentified factors that were not adjusted for in the analyses could also potentially influence our findings.
The present study contributes new insights by enhancing the current understanding of the potential impact of vitamin B 12 on male reproductive hormones in men with infertility.It provides a foundation for future investigations in the broader general population, in older age male cohorts, and for clinical trials exploring vitamin B 12 supplementation for men.The findings have potential clinical significance where the standard B 12 reference values may need to be re-examined to consider fertility.Our findings also suggest that B 12 concentrations may need to be assessed as part of routine measures in the management of male infertility.Enhanced knowledge in this field holds potential significant benefits, such as incorporating nutritional assessments and interventions with minimal side effects into male infertility management, including supporting more successful outcomes from assisted reproductive technologies.

TABLE 1
Subject characteristics.

TABLE 2
Spearman correlations for the independent association between serum vitamin B 12 concentration and reproductive hormone concentrations.

TABLE 3
Beta-coefficient (AE SE) and corresponding P for the linear association between serum vitamin B 12 concentration (pmol/L) and serum reproductive hormones concentrations.
The clinical status cutoffs were determined based on reproductive hormones outside of normal range: elevated FSH (>12.4IU/L),elevatedLH(>7.8IU/L), low TT (<9.2 nmol/L), and elevated estradiol (>146.8pmol/L).Serum reproductive hormones within the normal range and first tertile of serum vitamin B 12 concentration were used as the reference category for all analyses.2ORscomparetheodds of experiencing reproductive hormones outside of normal range for participants in the mid-tertile of serum vitamin B 12 concentration (>340 to <472.7 pmol/L) with participants in the lowest tertile (<340 pmol/L) of serum vitamin B 12 concentration.3ORscompare the odds of experiencing reproductive hormones outside of normal range for participants in the highest tertile (!472.7 pmol/L) of serum vitamin B 12 concentration with the odds for those in the lowest tertile of serum vitamin B 12 ( 340 pmol/L) concentration.