Abstract
This study aimed to analyze the associations between urinary genistein, daidzein, and equol concentrations and semen parameters in Japanese men exposed to high levels of isoflavone in their diet. Between September 2020 and March 2021, men seeking fertility treatment (n = 157) at Sanno Hospital (Tokyo, Japan) and Tsukuba Gakuen Hospital (Ibaraki, Japan) provided spot urine and semen specimens on the same day. Sperm concentration, sperm count, and total sperm motility were quantified. Urinary isoflavones were measured using liquid chromatography-tandem mass spectrometry and adjusted for specific gravity. The median (interquartile range [IQR]) total sperm count and concentration were 112 × 106 (50 × 106, 221 × 106) and 39 × 106 (17 × 106, 72 × 106) ml−1, respectively. Men in the second, third, and fourth quartile of urinary daidzein concentration had −40% (95% confidence interval [CI]: −59%, −13%), −37% (95% CI: −56%, −9%), and −32% (95% CI: −53%, −3%) low sperm count, respectively, than those in the lowest quartile. Men in the second, third, and fourth quartile of urinary genistein concentration had −23% (95% CI: −47%, 12%), −50% (95% CI: −66%, −26%), and −29% (95% CI: −51%, 3%) low sperm count than those in the lowest quartile, respectively. Sperm count showed no association with urinary equol concentration (P > 0.05). No associations were observed between urinary isoflavones and total sperm motility. A higher isoflavone intake may be associated with reduced sperm concentration and count. The effect of these alterations in semen parameters on the fecundity of couples trying to conceive remains unknown.
Keywords: Asia, daidzein, equol, genistein, semen
INTRODUCTION
Fertility rates remain below the population replacement level in many countries,1 and Japan is one of the countries with the lowest total fertility rate (TFR) worldwide. The country recorded a TFR of 1.20 in 2023, which is the lowest since 1947.2,3 Despite various policies implemented to increase fertility since the 1990s, the country continues to experience population aging and depopulation.4 The high prevalence of infertility among married couples is one of the factors contributing to the low fertility rates in the country. According to the National Fertility Survey in 2021, 39.2% of married women reportedly had infertility issues, and 22.6% had undergone tests or treatments for it.5 The 2019 report from the International Committee for Monitoring Assisted Reproductive Technologies revealed that among 3 411 005 reported cycles from 81 countries, 1 121 510 and 455 499 cycles were conducted in China and Japan, respectively.6 The reasons for the high utilization of fertility treatment need to be investigated from various viewpoints, including medical technology, the health insurance system, family norms, social expectations, and couples’ fecundity. Fecundity, referring to the ability to reproduce, can be quantified using fecundability, which is the monthly or cycle-specific probability of conception.7,8
Fecundity is affected by various personal behaviors, including diet.9,10,11,12 Regarding dietary factors, soy isoflavones are of interest, especially to Japanese, given their relatively high consumption levels.13,14 Previous studies have reported negative,15,16,17,18 positive,19 or no associations between soy isoflavone exposure and semen parameters.20,21,22 These studies differ in study design, exposure level, and method of exposure evaluation. The two intervention studies are from the USA22 and UK,20 where habitual isoflavone intake levels are much lower than those in Japan.23,24,25 In Japan, up to 2024, only one study has examined the association between isoflavone exposure and semen parameters, involving 42 married men seeking fertility consultation.16 Therefore, we aimed to analyze the relationship between isoflavone exposure and semen parameters in a larger sample of Japanese men.
PARTICIPANTS AND METHODS
Design and recruitment
This study utilized data and urine specimens obtained from the Interdisciplinary Investigation of Technology, Environment, and Fertility (IITEF) project (principal investigator: SK).26 Participants were men who sought fertility consultation and treatment at Sanno Hospital (Tokyo, Japan) from September 2020 to March 2021, and Tsukuba Gakuen Hospital (Ibaraki, Japan) from September 2020 to January 2021. Men aged between 20 years and 55 years who had never undergone vasectomy were eligible. Participants provided semen and urine specimens on the same day. Among 198 participants who submitted written informed consent, 177 provided additional consent to long-term storage of their urine and semen specimens for further studies. Information on age, height, weight, smoking status, and drinking habits was obtained using a self-administered questionnaire completed on the day of specimen collection. Information on left and right testis volume and varicocele status was obtained from medical records. The study protocol was approved by the Institutional Research Ethics Committees of the Graduate School of Medicine and Faculty of Medicine, University of Tokyo (Tokyo, Japan; Approval No. 2020057NI-[2]), Tsukuba Gakuen Hospital (Tsukuba, Japan; Approval No. 20–07), and the International University of Health and Welfare (Tokyo, Japan; Approval No. 20-S-8). The study was conducted in accordance with the principles of the Declaration of Helsinki 2024. Written informed consent and permission to publish the data were obtained from participants.
Semen analysis
The collection and analysis of semen samples followed the Semen Analyses Standardization Guidelines,27 with identical procedures at both hospitals, except for the method of measuring semen volume and the type of semen counting chamber. Participants collected semen samples by masturbation either at the hospitals or at home for convenience, without specific instructions on the number of abstinence days before collection. They were instructed to report the number of abstinence days and any missed ejaculate fractions. For semen specimens collected at home, participants were instructed to bring the samples to the hospitals within 60 min while maintaining them at the temperature between 20°C and 37°C. At the hospitals, semen specimens were kept at 37°C before and during the semen analysis. For specimens collected at the hospitals, liquefaction was checked within 30 min post-ejaculation, and analysis was conducted after liquefaction was completed. The volume of semen samples was assessed by weighing (in Tsukuba) or using a graduated syringe (in Tokyo). Sperm concentration and total motility were measured using the Sperm Motility and Morphology Analysis System with a computer-assisted semen analyzer (DITECT Co., Ltd., Tokyo, Japan), using either a Leja Standard Counting Chamber (Leja Products BV, Nieuw-Vennep, The Netherlands) in Ibaraki or a Makler counting chamber (Sefi-Medical Instruments Ltd., Haifa, Israel) in Tokyo. If a specimen was collected at home and showed abnormal motility, a retest was conducted using a specimen collected in the laboratory to determine whether the collection site influenced the results.
Serum hormone measurement
Venous blood samples were collected between 8:30 a.m. and 3:00 p.m. on or before the day of study enrollment as part of a standard clinical examination. Serum samples were sent to SRL Co., Ltd. (Tokyo, Japan) for measurement of follicle-stimulating hormone (FSH) and testosterone concentrations via chemiluminescent and electrochemiluminescence immunoassays, respectively.
Urinary isoflavone measurement
Urinary concentrations of genistein, daidzein, and equol were quantified using liquid chromatography-tandem mass spectrometry (LC/MS/MS) following solid-phase extraction (SPE) based on modified protocols from previous studies.28,29 A 20-μl urine sample was mixed with 180 μl of ultrapure water (1:10 dilution). After adding 10 ng of each isoflavone internal standard (isotope-labeled isoflavones: genistein-d4 and daidzein-d3 [Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA], and equol-d4 [Toronto Research Chemicals Inc., Toronto, Canada]), samples were hydrolyzed with 1.12 U of β-glucuronidase (from Escherichia coli K12; Roche, Penzberg, Germany) and 0.4 U of sulfatase (Type VIII, from abalone entrails; Sigma-Aldrich, St. Louis, MO, USA), then incubated overnight at 37°C using a thermomixer (Eppendorf SE, Hamburg, Germany). Samples were filtered using C18 SPE cartridges (Empore 4115SD-C18 [Octadecyl] Standard Density, 4 mm ml−1 Solid Phase Extraction Cartridges; CDS Analytical LLC, Oxford, PA, USA) and eluted with ethyl acetate/acetonitrile (1:1, v/v). The eluates were dried under nitrogen at 40°C, reconstituted with 200 μl of 50% methanol, and incubated for 10 min in an ultrasonic bath.
The prepared samples were analyzed using a tandem mass spectrometer (Xevo TQ MS; Waters Co., Milford, MA, USA) equipped with ultra-performance LC (ACQUITY UPLC; Waters Co.), using a reverse-phase (C18) column (1.7 µm, 2.1 mm × 150 mm, ACQUITY UPLC BEH C18; Waters Co.). Isoflavones were analyzed using electrospray ionization in negative mode. Calibration curves were constructed at concentrations of 0.1 ng ml−1, 1 ng ml−1, 10 ng ml−1, 100 ng ml−1, and 1000 ng ml−1 using native standards purchased from Sigma-Aldrich. Recovery rates were 91.0% for genistein (20 ng to 200 µl of 1:10 urine), 81.4% for daidzein (40 ng to 200 µl of 1:10 urine), and 104.2% for equol (4 ng to 200 µl of 1:10 urine). Detection limits (DL) were calculated using the formula:
DL = t(n−1, 0.05) × s × 2
where n = 5 (number of repeated measures), s is the standard deviation of repeated measurements (1 ng ml−1), and t(n−1, 0.05) is a t-value with n−1 degree of freedom and significance level of 0.05. The DLs were 0.005 μg ml−1 for genistein, 0.006 μg ml−1 for daidzein, and 0.010 μg ml−1 for equol. All samples had genistein and daidzein concentrations above the DLs, while 51.6% of the samples had equol concentrations above the DLs. For samples with equol concentrations lower than the DL of 0.010 μg ml−1, the value was calculated as DL/21/2 and used for statistical analyses. Inter-assay coefficients of variation (CVs), calculated using high and low isoflavone concentration quality controls, were 14.2% and 9.0% for genistein, 10.5% and 7.8% for daidzein, and 19.9% and 4.5% for equol (n = 5 for low and n = 6 for high concentrations), respectively. Intra-assay CVs were <7.1% for genistein, <4.0% for daidzein, and <8.3% for equol (n = 5, duplicate measurements).
Urinary isoflavone concentrations were adjusted for urine-specific gravity for statistical analyses, with non-adjusted and creatinine-adjusted isoflavone concentrations also presented. Methods of measuring urinary creatinine and specific gravity have been described previously.30
Statistical analyses
Statistical analyses were performed using R version 4.2.2.31 Spearman correlation coefficients were calculated among age, body mass index (BMI), abstinence days, right and left testis volumes, semen parameters, and urinary isoflavone concentrations. Two approaches were employed to examine the associations between urinary isoflavone and sperm parameters, which include (1) isoflavone (categorical) vs semen parameters (continuous) and (2) isoflavone (categorical) vs semen parameters (dichotomous).
Specific gravity-adjusted concentrations of urinary genistein and daidzein were categorized using quartiles. Urinary equol concentrations were categorized as non-excreters or excreters based on the classification method defined in a previous study.16 Specifically, participants with urinary equol concentration above the DL were categorized as excreters, whereas those with lower concentrations were categorized as non-excreters. Semen parameters were dichotomized into lower or higher groups using the reference values of 39 × 106 for sperm count, 16 × 106 ml−1 for sperm concentration, and 42% for total motility.32,33
Generalized linear models (GLMs) with a gamma distribution and a log link were used to analyze total sperm count and sperm concentration, with a value of 1 added to the original values to avoid zero values. For sperm concentration and total sperm count, regression coefficients and their 95% confidence intervals (CIs) were exponentiated and converted to percentage changes for figure presentation.15 For sperm motility, GLMs with a Gaussian distribution were used. Logistic regression models (GLMs with a binomial distribution) were used for dichotomous semen parameters.
The model included an outcome of continuous (sperm count, sperm concentration, or total motility) or dichotomous (lower sperm count, lower sperm concentration, or lower sperm motility) variables and an explanatory variable (urinary daidzein or genistein quartiles or equol excreter status), along with covariates associated with semen parameters in previous studies including age, abstinence days, mean testis volume, varicocele status (no, treated, or yes), BMI, smoking status (current, former, or never), and frequency of drinking alcohol (≤4 or >4 times a week).34,35,36,37,38,39,40,41 The mean testis volume was calculated as the mean of the left and right testis volumes. P < 0.05 was set as statistical significance.
Sensitivity analyses
The GLMs were analyzed using a subsample of participants who collected semen samples after 2–7 days of abstinence, following the 6th edition of World Health Organization criteria.33 Additional sensitivity analyses were conducted by adding serum testosterone and FSH as covariates in the GLMs.
Sample size calculation
A post hoc sample size calculation was performed using Pearson correlation for the association between sperm concentration and urinary daidzein concentration (r = −0.292, P = 0.064) based on a previous study in Japan,16 with α = 0.05 (both sides) and β = 0.20 (power = 0.80), and n was fixed as 90.42 Given this calculation, the available sample size was deemed sufficient for our study’s objective.
RESULTS
Urinary isoflavone concentrations were quantified for urine samples from 177 participants who consented to the long-term storage of their biological specimens. After excluding 18 men with unknown abstinence days and two men with unknown testis volume, the final analytic cohort comprised 157 participants. The basic characteristics of the samples analyzed are summarized in Table 1. Proportions below the reference values32,33 were 22.3% for sperm concentration, 20.4% for total sperm count, and 52.9% for sperm motility (Table 2). Urinary daidzein and genistein concentrations were highly positively correlated (rho = 0.892, P < 0.001; Supplementary Table 1). Daidzein and genistein concentrations showed significant and non-significant negative correlations with total sperm count, total motility, and sperm concentration (Supplementary Table 1). Scatter plots of urinary isoflavone vs total sperm count (Supplementary Figure 1 (120.6KB, tif) ), sperm concentration (Supplementary Figure 2 (112KB, tif) ), and total motility (Supplementary Figure 3 (121.8KB, tif) ) are provided. Individual variations of urinary isoflavone concentrations are large, and no clear correlations are visible from these scatter plots. Although all the urine samples contained genistein and daidzein concentrations above the DLs, only 51.6% contained equol concentrations above the DL of 0.010 μg ml−1 (Table 3). Therefore, based on our criteria, 51.6% were categorized as equol excreters, while 48.4% were categorized as non-excreters.
Table 1.
Demographic, anthropometric, and lifestyle characteristics of 157 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| Characteristic | Value |
|---|---|
| Age (year), mean (s.d.) | 37 (6) |
| Height (cm), mean (s.d.) | 173 (6) |
| Weight (kg), mean (s.d.) | 71 (12) |
| BMI (kg m−2), mean (s.d.) | 23.7 (3.5) |
| Abstinence time (day), median (range) | 3 (0–14) |
| Left testis volume (ml), mean (s.d.) | 19.4 (4.7) |
| Right testis volume (ml), mean (s.d.) | 20.4 (4.6) |
| Varicocele, n (%) | |
| None | 52 (33.1) |
| Treated | 50 (31.8) |
| Current | 55 (35.0) |
| Smoking, n (%) | |
| Current | 25 (15.9) |
| Former | 47 (29.9) |
| Never | 85 (54.1) |
| Alcohol consumption, n (%) | |
| Once per week or less | 97 (61.8) |
| More than once per week | 60 (38.2) |
BMI: body mass index; s.d.: standard deviation
Table 2.
Semen parameters of 157 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| Semen parameter | Proportion below cutoffa, n (%) | Mean (s.d.) | Percentile | ||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| 5th | 25th | 50th | 75th | 95th | |||
| Semen volume (ml) | NA | 3.4 (1.5) | 1.1 | 2.2 | 3.3 | 4.4 | 5.7 |
| Sperm concentration (×106 ml−1) | 35 (22.3) | 51 (46) | 3 | 17 | 39 | 72 | 129 |
| Total sperm count (×106) | 32 (20.4) | 158 (137) | 7 | 50 | 112 | 221 | 427 |
| Sperm motility (%) | 83 (52.9) | 41.9 (19.8) | 10.6 | 28.0 | 41.0 | 56.4 | 72.2 |
| Testosteroneb (ng dl−1) | NA | 499.2 (241.5) | 186.4 | 365.1 | 465.0 | 582.0 | 850.0 |
| FSHc (mIU ml−1) | NA | 5.2 (3.8) | 2.0 | 3.0 | 4.2 | 6.6 | 11.3 |
aSperm concentration <16×106 ml−1, total sperm count <39×106, and sperm motility <42%; bn=142; cn=143. FSH: follicle-stimulating hormone; s.d.: standard deviation; NA: not available
Supplementary Table 1.
Spearman correlation coefficients between age, body mass index, abstinence days, testis volume, semen parameters, urinary isoflavones, and serum testosterone and follicle-stimulating hormone concentrations of 157 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1. Age | 0.213** | −0.034 | 0.217** | −0.370*** | −0.036 | −0.195* | −0.231** | 0.199* | 0.013 | 0.040 | −0.245** | 0.164 |
| 2. BMI | 0.000 | 0.206** | −0.243** | −0.048 | −0.141 | −0.135 | −0.002 | 0.017 | −0.020 | −0.438*** | 0.086 | |
| 3. Abstinence days | −0.101 | 0.232** | 0.203* | −0.068 | 0.287*** | 0.030 | −0.001 | −0.014 | 0.104 | −0.026 | ||
| 4. Mean testis volume | −0.061 | 0.294*** | 0.091 | 0.249** | 0.009 | −0.071 | −0.118 | −0.140 | −0.244** | |||
| 5. Semen volume | −0.117 | −0.043 | 0.321*** | 0.018 | −0.073 | −0.130 | 0.271** | −0.163 | ||||
| 6. Sperm concentration | 0.422*** | 0.878*** | 0.027 | −0.140 | −0.136 | 0.073 | −0.321*** | |||||
| 7. Total motility | 0.353*** | −0.031 | −0.142 | −0.144 | −0.034 | −0.106 | ||||||
| 8. Total sperm count | 0.027 | −0.181* | −0.200* | 0.206* | −0.412*** | |||||||
| 9. Equol† | −0.055 | 0.028 | 0.062 | −0.016 | ||||||||
| 10. Daidzein† | 0.892*** | −0.046 | 0.159 | |||||||||
| 11. Genistein† | 0.015 | 0.136 | ||||||||||
| 12. Testosterone | 0.031 |
*P<0.05; **P<0.01; ***P<0.001; †Urinary concentrations, specific gravity corrected. BMI: body mass index
Table 3.
Distributions of urinary isoflavone concentrations among 157 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| Phytoestrogen | Percentage above detection limitb (%) | Geometric, mean (s.d.) | Percentile | ||||
|---|---|---|---|---|---|---|---|
|
| |||||||
| 5th | 25th | 50th | 75th | 95th | |||
| Genistein (µg ml−1) | 100.0 | 0.57 (4.38) | 0.06 | 0.21 | 0.50 | 1.94 | 5.49 |
| Genisteina (µg ml−1) | 100.0 | 0.62 (4.09) | 0.06 | 0.23 | 0.60 | 1.98 | 5.36 |
| Genistein (µg per mg creatinine) | 100.0 | 0.54 (4.12) | 0.06 | 0.20 | 0.55 | 1.42 | 5.36 |
| Daidzein (µg ml−1) | 100.0 | 1.24 (4.87) | 0.08 | 0.44 | 1.19 | 4.08 | 15.0 |
| Daidzeina (µg ml−1) | 100.0 | 1.35 (4.57) | 0.10 | 0.51 | 1.45 | 4.50 | 13.8 |
| Daidzein (µg per mg creatinine) | 100.0 | 1.18 (4.61) | 0.08 | 0.46 | 1.24 | 3.49 | 11.4 |
| Equol (µg ml−1) | 51.6 | 0.05 (11.06) | <0.01 | <0.01 | 0.01 | 0.32 | 6.02 |
| Equola (µg ml−1) | 51.6 | 0.06 (11.44) | <0.01 | <0.01 | 0.02 | 0.39 | 5.17 |
| Equol (µg per mg creatinine) | 51.6 | 0.05 (11.47) | <0.01 | <0.01 | 0.01 | 0.32 | 4.63 |
aAdjusted for specific gravity of urine; bgenistein 0.005 µg ml−1; daidzein 0.006 µg ml−1; equol 0.010 µg ml−1. For equol concentrations below the detection limit, 0.007 µg ml−1 was substituted. s.d.: standard deviation
Men in the second (exponentiated coefficient: 0.60, 95% CI: 0.41, 0.87), third (exponentiated coefficient: 0.63, 95% CI: 0.44, 0.91), and fourth (exponentiated coefficient: 0.68, 95% CI: 0.47, 0.97) quartiles had total sperm count lower than that in the first quartile of urinary daidzein concentrations. Urinary genistein quartiles showed non-linear associations with total sperm count (Figure 1 and Supplementary Table 2). Less consistent associations were observed between sperm concentration and genistein and daidzein quartiles (Figure 1 and Supplementary Table 3). Sperm motility showed no clear associations with any of the isoflavones measured (Figure 1 and Supplementary Table 4). Equol excreter status was not associated with any of the semen parameters examined. Smoking status was not significantly associated with total sperm count, sperm concentration, or total motility (Supplementary Table 2–4). A greater testis volume was associated with a higher total sperm count and concentration, while having varicocele and a higher BMI were associated with a lower total sperm count and concentration (Supplementary Table 2 and 3). Neither testis volume nor BMI was associated with sperm motility (Supplementary Table 4).
Figure 1.
Percentage changes or coefficients and 95% CIs for semen parameters associated with urinary isoflavone quartiles. In each model, adjustments were made for age, abstinence days, mean testis volume, varicocele status, BMI, smoking status, and frequency of drinking alcohol. BMI: body mass index; CI: confidence intervals; DAI: daidzein; EQU: equol; GEN: genistein; E: excreter; N: non-excreter; Q1–4: quartile 1–4.
Supplementary Table 2.
Associations between total sperm count and categories of urinary isoflavone concentrations and other individual characteristics of 157 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| Model 1 | Model 2 | Model 3 | |
|---|---|---|---|
| Age | 0.97** (0.95, 0.99) | 0.96** (0.94, 0.99) | 0.97* (0.95, 0.99) |
| Abstinence days | 1.12*** (1.05, 1.19) | 1.11** (1.04, 1.18) | 1.13*** (1.05, 1.20) |
| Mean testis volume | 1.08*** (1.05, 1.11) | 1.08*** (1.04, 1.11) | 1.08*** (1.05, 1.11) |
| Varicocele (reference: no) | 1.00 | 1.00 | 1.00 |
| Treated | 0.92 (0.67, 1.28) | 0.86 (0.62, 1.18) | 0.82 (0.59, 1.15) |
| Yes | 0.67* (0.49, 0.93) | 0.69* (0.50, 0.96) | 0.72 (0.52, 1.00) |
| BMI | 0.95* (0.92, 0.99) | 0.96* (0.93, 1.00) | 0.96* (0.92, 0.99) |
| Smoking (reference: current) | 1.00 | 1.00 | 1.00 |
| Former | 1.13 (0.74, 1.72) | 1.14 (0.74, 1.77) | 1.25 (0.80, 1.94) |
| Never | 1.11 (0.77, 1.62) | 1.13 (0.76, 1.66) | 1.26 (0.85, 1.87) |
| Drinking alcohol (ref: 4 or less times a week) | 1.00 | 1.00 | 1.00 |
| >4 times a week | 0.92 (0.70, 1.22) | 0.92 (0.70, 1.22) | 0.90 (0.67, 1.21) |
| Urinary genistein (reference: Q1) | |||
| Q2 | 0.77 (0.53, 1.12) | ||
| Q3 | 0.50*** (0.34, 0.74) | ||
| Q4 | 0.71 (0.49, 1.03) | ||
| Urinary daidzein (reference: Q1) | |||
| Q2 | 0.60** (0.41, 0.87) | ||
| Q3 | 0.63* (0.44, 0.91) | ||
| Q4 | 0.68* (0.47, 0.97) | ||
| Urinary equol (reference: non-excreter) | |||
| Excreter | 0.94 (0.71, 1.24) | ||
| n | 157 | 157 | 157 |
| AIC | 1881.90 | 1884.59 | 1888.98 |
| BIC | 1924.69 | 1927.38 | 1925.66 |
| Pseudo R2 | 0.29 | 0.27 | 0.23 |
*P<0.05; **P<0.01; ***P<0.001; Exponentiated coefficients and their 95% CIs are shown for each GLM with a Poisson distribution. CIs: confidence intervals; GLM: generalized linear model; BMI: body mass index
Supplementary Table 3.
Associations between sperm concentration and categories of urinary isoflavone concentrations and other individual characteristics of 157 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| Model 1 | Model 2 | Model 3 | |
|---|---|---|---|
| Age | 1.01 (0.98, 1.03) | 1.00 (0.98, 1.03) | 1.01 (0.98, 1.03) |
| Abstinence days | 1.07 (1.00, 1.14) | 1.06 (0.99, 1.13) | 1.07 (1.00, 1.15) |
| Mean testis volume | 1.06*** (1.03, 1.10) | 1.06*** (1.03, 1.09) | 1.06*** (1.03, 1.10) |
| Varicocele (reference: no) | 1.00 | 1.00 | 1.00 |
| Treated | 0.83 (0.59, 1.18) | 0.84 (0.59, 1.18) | 0.81 (0.57, 1.14) |
| Yes | 0.67* (0.47, 0.94) | 0.68* (0.48, 0.96) | 0.69* (0.49, 0.97) |
| BMI | 0.96 (0.93, 1.00) | 0.97 (0.93, 1.01) | 0.97 (0.93, 1.01) |
| Smoking (reference: current) | 1.00 | 1.00 | 1.00 |
| Former | 1.05 (0.67, 1.65) | 1.06 (0.66, 1.68) | 1.12 (0.71, 1.76) |
| Never | 1.04 (0.70, 1.56) | 1.07 (0.71, 1.62) | 1.15 (0.77, 1.72) |
| Drinking alcohol (reference: 4 or less times a week) | 1.00 | 1.00 | 1.00 |
| >4 times a week | 1.06 (0.78, 1.43) | 1.05 (0.78, 1.42) | 1.02 (0.75, 1.38) |
| Urinary genistein (reference: Q1) | |||
| Q2 | 1.07 (0.72, 1.60) | ||
| Q3 | 0.75 (0.50, 1.13) | ||
| Q4 | 0.85 (0.57, 1.27) | ||
| Urinary daidzein (reference: Q1) | |||
| Q2 | 0.68 (0.46, 1.01) | ||
| Q3 | 0.75 (0.51, 1.12) | ||
| Q4 | 0.71 (0.48, 1.04) | ||
| Urinary equol (reference: non-excreter) | |||
| Excreter | 0.92 (0.69, 1.22) | ||
| n | 157 | 157 | 157 |
| AIC | 1547.28 | 1546.39 | 1547.03 |
| BIC | 1590.07 | 1589.17 | 1583.70 |
| Pseudo R2 | 0.18 | 0.18 | 0.16 |
*P<0.05; ***P<0.001; Exponentiated coefficients and their 95% CIs from GLMs assuming a Poisson distribution are shown. CIs: confidence intervals; GLMs: generalized linear models; BMI: body mass index
Supplementary Table 4.
Associations between total sperm motility and categories of urinary isoflavone concentrations and other individual characteristics of 157 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| Genistein | Daidzein | Equol | |
|---|---|---|---|
| Age | −0.66* (−1.18, −0.13) | −0.66* (−1.18, −0.14) | −0.62* (−1.14, −0.09) |
| Abstinence days | 0.26 (−1.26, 1.78) | 0.35 (−1.16, 1.87) | 0.35 (−1.15, 1.85) |
| Mean testis volume | 0.61 (−0.12, 1.34) | 0.65 (−0.06, 1.36) | 0.71 (0.00, 1.41) |
| Varicocele (reference: no) | |||
| Treated | −2.77 (−10.6, 5.07) | −2.49 (−10.2, 5.16) | −3.73 (−11.3, 3.85) |
| Yes | −5.76 (−13.4, 1.89) | −5.62 (−13.3, 2.00) | −5.16 (−12.7, 2.36) |
| BMI | −0.66 (−1.58, 0.25) | −0.63 (−1.54, 0.28) | −0.72 (−1.62, 0.17) |
| Smoking (reference: current) | |||
| Former | 5.75 (−4.35, 15.9) | 6.72 (−3.66, 17.1) | 6.16 (−3.81, 16.1) |
| Never | 2.49 (−6.51, 11.5) | 3.41 (−5.85, 12.7) | 3.23 (−5.65, 12.1) |
| Drinking alcohol (reference: 4 or less times a week) | |||
| >4 times a week | 6.21 (−0.53, 13.0) | 6.02 (−0.71, 12.8) | 6.24 (−0.42, 12.9) |
| Urinary genistein (reference: Q1) | |||
| Q2 | −3.03 (−12.0, 5.92) | ||
| Q3 | −1.52 (−10.7, 7.64) | ||
| Q4 | −4.79 (−13.7, 4.11) | ||
| Urinary daidzein (reference: Q1) | |||
| Q2 | −3.11 (−12.0, 5.81) | ||
| Q3 | 0.42 (−8.35, 9.19) | ||
| Q4 | −4.61 (−13.2, 4.03) | ||
| Urinary equol (reference: non-excreter) | |||
| Excreter | −3.63 (−9.91, 2.65) | ||
| n | 157 | 157 | 157 |
| AIC | 1387.06 | 1386.47 | 1383.02 |
| BIC | 1429.85 | 1429.26 | 1419.69 |
| Pseudo R2 | 0.13 | 0.14 | 0.13 |
*P<0.05; Coefficients and their 95% CIs from GLMs assuming a Gaussian distribution are shown. CIs: confidence intervals; GLMs: generalized linear models
The overall associations between semen parameters and urinary isoflavone levels remained consistent in the sensitivity analyses with a limited sample size involving participants with 2–7 abstinence days (Supplementary Table 5). Further adjustment with serum testosterone and FSH concentrations did not notably alter these associations (Supplementary Table 6).
Supplementary Table 5.
Associations between semen parameters and categories of urinary isoflavone concentrations of 143 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| Total sperm count | Sperm concentration | Total sperm motility | ||||
|---|---|---|---|---|---|---|
|
|
|
|
||||
| Exponentiated coefficients (95% CI) | P | Exponentiated coefficients (95% CI) | P | Coefficients (95% CI) | P | |
| Genistein 1Q | Reference | Reference | Reference | |||
| 2Q | 0.79 (0.54, 1.16) | 0.228 | 1.08 (0.71, 1.65) | 0.720 | −1.69 (−10.7, 7.33) | 0.713 |
| 3Q | 0.51 (0.35, 0.77) | 0.001 | 0.72 (0.46, 1.12) | 0.146 | −0.63 (−10.1, 8.82) | 0.897 |
| 4Q | 0.66 (0.45, 0.98) | 0.042 | 0.79 (0.51, 1.21) | 0.274 | −6.00 (−15.2, 3.23) | 0.205 |
| Daidzein 1Q | Reference | Reference | Reference | |||
| 2Q | 0.56 (0.38, 0.82) | 0.003 | 0.61 (0.4, 0.94) | 0.026 | −5.41 (−14.5, 3.66) | 0.245 |
| 3Q | 0.63 (0.43, 0.92) | 0.018 | 0.70 (0.46, 1.07) | 0.105 | −0.28 (−9.37, 8.80) | 0.952 |
| 4Q | 0.60 (0.41, 0.88) | 0.009 | 0.62 (0.41, 0.94) | 0.027 | −6.94 (−15.9, 1.99) | 0.130 |
| Equol non-excreter | Reference | Reference | Reference | |||
| Excreter | 0.88 (0.66, 1.17) | 0.389 | 0.91 (0.67, 1.23) | 0.538 | −3.87 (−10.3, 2.59) | 0.243 |
The sample is limited to those with 2–7 abstinence days. All the models were adjusted for age, abstinence days, mean testicular volume, varicocele status, BMI, smoking, and drinking. CI: confidence interval
Supplementary Table 6.
Associations between semen parameters and categories of urinary isoflavone concentrations of 142 men in the Interdisciplinary Investigation of Technology, Environment, and Fertility study, 2020–2021
| Total sperm count | Sperm concentration | Total sperm motility | ||||
|---|---|---|---|---|---|---|
|
|
|
|
||||
| Exponentiated coefficients (95% CI) | P | Exponentiated coefficients (95% CI) | P | Coefficients (95% CI) | P | |
| Genistein 1Q | Reference | Reference | Reference | |||
| 2Q | 0.68 (0.48, 0.97) | 0.033 | 1.05 (0.70, 1.58) | 0.820 | −5.32 (−14.51, 3.87) | 0.259 |
| 3Q | 0.52 (0.37, 0.75) | 0.001 | 0.81 (0.54, 1.23) | 0.333 | −0.69 (−9.99, 8.61) | 0.884 |
| 4Q | 0.75 (0.53, 1.07) | 0.119 | 0.95 (0.63, 1.44) | 0.826 | −5.69 (−14.9, 3.55) | 0.230 |
| Daidzein 1Q | Reference | Reference | Reference | |||
| 2Q | 0.7 (0.49, 0.99) | 0.048 | 0.81 (0.54, 1.24) | 0.337 | −4.88 (−14.1, 4.31) | 0.300 |
| 3Q | 0.64 (0.45, 0.91) | 0.014 | 0.82 (0.54, 1.23) | 0.333 | 0.31 (−8.69, 9.31) | 0.946 |
| 4Q | 0.77 (0.54, 1.09) | 0.143 | 0.83 (0.55, 1.25) | 0.375 | −5.73 (−14.7, 3.23) | 0.212 |
| Equol non-excreter | Reference | Reference | Reference | |||
| Excreter | 0.93 (0.72, 1.20) | 0.559 | 0.88 (0.66, 1.18) | 0.406 | −2.41 (−8.92, 4.10) | 0.469 |
All the models were adjusted for age, abstinence days, mean testicular volume, varicocele status, BMI, smoking, drinking, and serum testosterone and FSH levels. CI: confidence interval; FSH: follicle-stimulating hormone; BMI: body mass index
The associations were different when dichotomized semen parameters were used as outcomes (Table 4). No associations were observed between categories of urinary isoflavone concentrations and any of the dichotomized categories of total sperm count, sperm concentration, or sperm motility.
Table 4.
Odds of having lower sperm parameters by quartiles of genistein and daidzein and equol excreter status
| Urinary isoflavone concentration quartiles | Total sperm count <39×106 | Sperm concentration <16×106 ml−1 | Total motility <42% | |||
|---|---|---|---|---|---|---|
|
|
|
|
||||
| OR (95% CI) | P | OR (95% CI) | P | OR (95% CI) | P | |
| Genistein | ||||||
| Q1 | Reference | Reference | Reference | |||
| Q2 | 1.84 (0.41, 8.23) | 0.424 | 2.83 (0.72, 11.2) | 0.137 | 2.16 (0.8, 5.80) | 0.127 |
| Q3 | 1.69 (0.35, 8.25) | 0.518 | 1.62 (0.37, 7.00) | 0.518 | 1.24 (0.45, 3.40) | 0.673 |
| Q4 | 3.04 (0.69, 13.3) | 0.141 | 1.57 (0.38, 6.56) | 0.537 | 2.26 (0.85, 6.02) | 0.104 |
| Daidzein | ||||||
| Q1 | Reference | Reference | Reference | |||
| Q2 | 0.84 (0.20, 3.64) | 0.819 | 1.65 (0.45, 6.11) | 0.450 | 0.86 (0.32, 2.27) | 0.756 |
| Q3 | 0.84 (0.20, 3.62) | 0.819 | 1.18 (0.31, 4.46) | 0.811 | 0.88 (0.34, 2.27) | 0.789 |
| Q4 | 2.30 (0.58, 9.07) | 0.234 | 1.09 (0.28, 4.19) | 0.902 | 2.22 (0.85, 5.83) | 0.105 |
| Equol | ||||||
| Non-excreter | Reference | Reference | Reference | |||
| Excreter | 0.57 (0.21, 1.54) | 0.270 | 1.13 (0.45, 2.83) | 0.795 | 1.16 (0.58, 2.31) | 0.667 |
Each logistic regression model includes one of the dichotomized sperm parameters and one of the urinary isoflavones. All the models were adjusted for age, abstinence days, mean testicular volume, varicocele status, BMI, smoking, and drinking. BMI: body mass index; CI: confidence interval; OR: odds ratio; Q1–4: quartile 1–4
DISCUSSION
Urinary concentrations of daidzein and genistein were negatively associated with sperm count and concentration. None of the isoflavones was associated with the likelihood of having semen parameters below the reference values.32,33 Urinary equol concentration showed no association with sperm count, sperm concentration, or total motility.
In our findings, the negative association between isoflavone and semen parameters aligns with that of previous studies in Japan16 and China,15 although some differences exist. A Japanese study of 42 men seeking fertility treatment found a negative correlation between urinary daidzein concentration and sperm concentration. Men with detectable urinary equol concentrations ≥0.005 μg ml−1 had lower sperm motility than those with undetectable equol concentrations (standardized coefficient = −0.315, P < 0.05),16 which is inconsistent with our findings. In the Japanese study,16 the association between equol detectability and sperm motility was found in a multiple regression analysis, which was adjusted for smoking, caffeine consumption, and urinary concentrations of pyrethroid insecticide (3-phenoxybenzoic acid). Urinary genistein concentration was not quantified, and the duration of sexual abstinence ranged between 1 day and 7 days in the Japanese study,16 whereas it ranged between 0 and 14 days in our study. In the previous Japanese study,16 compared to non-smokers, current smokers showed higher sperm motility, but no such association was observed in our study. The exact mechanisms underlying the contrasting findings between the two studies are unclear. Future studies could evaluate the equol-producing ability and its relation to sperm count, concentration, and motility by administering known amounts of daidzein to participants. In a Chinese study of 1319 reproductive-age men, nine phytoestrogens, including genistein, daidzein, and equol, were quantified in semen samples. Men with higher seminal genistein concentrations had lower sperm concentrations and counts; however, no associations were observed between seminal daidzein and lower sperm concentrations or counts.15 The Chinese study differs from our study in several aspects, including the use of different biological specimens to evaluate isoflavone exposure. Specifically, men with higher seminal equol concentrations had lower sperm concentrations and counts, while those in the highest quartile of seminal daidzein concentration had higher total motility than those in the lower quartiles. Smoking status was adjusted in the regression models in the Chinese study,15 but the coefficients were not shown. No associations between smoking status and semen parameters in our study contrasts with the findings from review studies that reported negative impact of smoking on sperm concentration and motility.36,37 Heterogeneity of the association between smoking and semen parameters exists,36 and the source of heterogeneity needs to be investigated in future studies.
Isoflavone exposure may adversely affect spermatogenesis via genomic and non-genomic pathways.43 However, it remains unclear whether higher isoflavone concentrations in urine indicate greater bioavailability in the target organs. The bioavailability of isoflavones depends on their chemical forms (aglycone or glucoside), metabolism by both intestinal and microbiome enzymes, absorption in the intestine, and distribution to target organs.43 In our study, we observed negative associations between sperm count and concentration and urinary daidzein and genistein concentrations, indicating that urinary concentrations partly reflect bioavailable isoflavones in target organs, such as the testis. Consequently, isoflavone intake may adversely affect sperm production in men. However, these adverse effects may be exerted via mechanisms other than decreased testosterone or increased FSH because adjusting for these hormones in our analysis did not alter the observed associations (Supplementary Table 6).
Furthermore, our findings contrast with intervention studies in the UK20 and USA,22 which found no effects of isoflavone intake on semen parameters. In the UK study, daily supplementation with 40 mg of isoflavones for 2 months did not change testicular volume or semen parameters.20 The American study was a randomized crossover intervention, and participants ingested low-isoflavone soy (1.64 mg isoflavones per day), high-isoflavone soy (61.7 mg isoflavones per day), or milk protein isolates. None of the semen parameters examined were affected by the low- or high-isoflavone interventions.22 These isoflavone intake levels seem to be similar to the levels consumed by Japanese adults. Dietary records in Japan estimate that the mean genistein intake is 23.36 mg per day, and daidzein intake averages 14.52 mg per day.14
The discrepancy between these Asian and Western studies may be due to the differences in habitual consumption of isoflavones. We suspect that the reproductive impact of isoflavones may be more evident in a population with a high isoflavone intake than that in a population with a low isoflavone intake. Urinary genistein and daidzein concentrations in our study were higher than those previously reported in the USA21,44 and China,17 but similar to those in Japan.13,16 This suggests that the general Japanese population, without any interventions, consumes more isoflavones than Americans or Chinese. In a nationally representative sample of American men, the median urinary concentrations of genistein and daidzein were 0.031 μg ml−1 and 0.079 μg ml−1, respectively.44 The sample in our study showed higher median concentrations at 0.501 μg ml−1 for genistein and 1.19 μg ml−1 for daidzein. In the USA, for male partners of couples seeking pregnancy, the geometric mean of urinary genistein and daidzein concentrations were 0.041 μg ml−1 (151 nmol l−1) and 0.087 μg ml−1 (342 nmol l−1), respectively.21 In infertile men with at least one abnormal semen parameter in China, the geometric mean of urinary genistein and daidzein concentrations were 56.11 μg per g creatinine and 64.16 μg per g creatinine, respectively.17 In our study samples, the geometric mean of urinary genistein and daidzein concentrations were 538 μg per g creatinine and 1178 μg per g creatinine, respectively. Although the sample sizes were limited, reported urinary genistein and daidzein concentrations in Japan13 are similar to those observed in our study. Kunisue et al.13 reported median concentrations of 0.380 μg ml−1 for urinary genistein and 1.5 μg ml−1 for daidzein in a sample of 15 men from Ehime prefecture, Japan. Among the male partners of couples seeking fertility treatment in Japan, the median concentration of urinary daidzein, adjusted to the specific gravity of 1.010, was 1.14 μg ml−1.16
Although the estrogenic activities of genistein and equol are much higher than that of daidzein,45 the effects of genistein and daidzein did not differ in relation to sperm count and concentration in our study. This may be because dietary sources for both isoflavones are common, and their urinary concentrations are highly positively correlated (rho = 0.892, P < 0.001). We speculate that the high positive correlation is a result of dietary coingestion. According to a survey utilizing dietary records in Japan, tofu, miso, and natto (fermented beans) contributed to 41.8%, 26.4%, and 16.5%, respectively, of total daidzein intake, and 42.2%, 22.7%, and 17.0%, respectively, of total genistein intake.14 Considering that dietary sources of both isoflavones are largely overlapping, for evaluating the effects of genistein and daidzein separately, an intervention study with controlled diet will be needed. Urinary concentrations of daidzein and equol were not correlated, reflecting interindividual variations in metabolism.46 Toshima et al.16 reported lower total motility in equol excreters than in non-excreters; however, our study found no association between equol concentrations and any semen parameters examined. The differences in the DLs for equol, 0.005 μg ml−1,16 and 0.010 μg ml−1 in this study, do not explain the discrepancy. Further studies are needed to ascertain whether equol excreter status affects semen parameters.
Despite these findings, it remains unclear whether the observed differences in sperm count or concentration by urinary isoflavone levels could contribute to differences in a couple’s fecundity. The reference values32 used in this study are based on the fifth percentiles of semen parameters in fertile men. Campbell et al.32 stated that these reference values cannot be used to categorize men into fertile and infertile groups. Although a higher total motile sperm count is associated with a higher pregnancy rate with intrauterine insemination,47 in subfertile couples attempting to conceive naturally, neither sperm motility nor morphology predicted natural conception rates.48 Overall, the observed negative associations between isoflavones and sperm count and concentration in our study are insufficient to support assumptions about the fertility-decreasing effects of isoflavone intake. Future studies should target general populations and multiple exposures to elucidate the combined impacts of environmental exposures on fecundity and fertility. Although an American study49 reported no association between men’s urinary isoflavone concentration and fecundity, a comparable study has not been conducted in any setting with high habitual isoflavone intake. It is worth investigating whether subtle alterations in semen parameters, like those observed in this study, can affect a couple’s fecundity. Furthermore, considering the combined effects of exposure to multiple chemicals is essential.
This study has some limitations. Although we targeted men who consulted a doctor regarding infertility, we were unable to obtain information on the underlying causes of infertility. The participants included men with infertility issues and men whose partners had infertility issues. The heterogeneity of the sample made it difficult to interpret the findings. The observed association between urinary isoflavones and semen parameters may differ in a general Japanese population. The use of a single spot urine and a single semen test may have weakened the associations between them due to within-person variability in isoflavone intake and excretion and semen parameters. Moreover, it is unclear to what extent urinary isoflavone concentrations reflect bioavailable isoflavones in the testis. Blood was collected up to 1757 days before the study, which may have masked the true association between isoflavone exposure and hormone levels. Furthermore, abstinence days ranged between 0 day and 14 days, although the sensitivity analyses using samples with 2–7 abstinence days revealed similar results as the main analyses.
In summary, we found negative associations between urine isoflavone concentration with sperm count and concentration, but not with sperm motility, among male partners of infertile couples in Japan. In contrast, urinary isoflavone concentrations did not correlate with the likelihood of lower sperm count or motility defined by commonly used reference values, which are based on the 5th percentile values of semen parameters of fertile men from twelve countries.32,33 It remains unknown whether alterations in sperm count, concentration, or motility beyond the reference values affect a couple’s fecundity. Besides these parameters, frequency of insemination, quality of egg, and many other factors contribute to variability in fecundity across and within couples.50 Further research is needed to examine the potential impact of diet and other personal behaviors on semen parameters and a couple’s fecundity and fertility.
AUTHOR CONTRIBUTIONS
SK contributed to the conceptualization of the study, participated in the investigation, curated and analyzed the data, wrote and revised the manuscript and created the visualizations, performed project administration, and acquired funding. YM conceptualized the methodology, participated in the investigation, and helped review and edit the manuscript. KY participated in the investigation, helped review and edit the manuscript, and supervised the study. MU participated in the investigation and helped review and edit the manuscript. TI contributed to the conceptualization of the study, participated in the investigation, provided resources, helped review and edit the manuscript, and supervised the study. All authors read and approved the final manuscript and agreed to be accountable for all aspects of the work.
COMPETING INTERESTS
All authors declare no competing interests.
Scatter plots of total sperm count and urinary (a) genistein, (b) daidzein, and (c) equol concentrations. Urinary equol concentrations below the detection limit of 0.010 μg/mL were substituted with 0.007 μg/mL. The dashed horizontal lines indicate the reference value of 39 million.
Scatter plots of sperm concentration and urinary (a) genistein, (b) daidzein, and (c) equol concentrations. Urinary equol concentrations below the detection limit of 0.010 μg/mL were substituted with 0.007 μg/mL. The dashed horizontal lines indicate the reference value of 16 million/mL.
Scatter plots of total sperm motility and urinary (a) genistein, (b) daidzein, and (c) equol concentrations. Urinary equol concentrations below the detection limit of 0.010 μg/mL were substituted with 0.007 μg/mL. The dashed horizontal lines indicate the reference value of 42%.
ACKNOWLEDGMENTS
The authors thank all the participants and hospital staff members for cooperating in the study. We thank Mr. Tatsuji Ihana (Sanno Hospital, Tokyo, Japan) and Ms. Fumiko Kariya (The University of Tokyo, Tokyo, Japan) for helping with the data collection and Prof. Jun Yoshinaga (Toyo University, Asaka, Japan) for commenting on the manuscript. We also thank Prof. Masatoshi Nakajima (The University of Tokyo, Tokyo, Japan) and Prof. Keitaro Tanoi (The University of Tokyo, Tokyo, Japan) for their support in the UPLC-MS/MS measurement. This work was supported by the Grant for Environmental Research Projects from The Sumitomo Foundation and by the Japan Society for the Promotion of Science (JSPS) Topic-Setting Program to Advance Cutting-Edge Humanities and Social Sciences Research, Global Initiatives (grant No. JSPS00119217822; PI: SK). The funding agency had no role in the study design; in the collection, analysis, and interpretation of the data; in the writing of the report; and in the decision to submit the article for publication.
Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.
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Supplementary Materials
Scatter plots of total sperm count and urinary (a) genistein, (b) daidzein, and (c) equol concentrations. Urinary equol concentrations below the detection limit of 0.010 μg/mL were substituted with 0.007 μg/mL. The dashed horizontal lines indicate the reference value of 39 million.
Scatter plots of sperm concentration and urinary (a) genistein, (b) daidzein, and (c) equol concentrations. Urinary equol concentrations below the detection limit of 0.010 μg/mL were substituted with 0.007 μg/mL. The dashed horizontal lines indicate the reference value of 16 million/mL.
Scatter plots of total sperm motility and urinary (a) genistein, (b) daidzein, and (c) equol concentrations. Urinary equol concentrations below the detection limit of 0.010 μg/mL were substituted with 0.007 μg/mL. The dashed horizontal lines indicate the reference value of 42%.