Association between nutrient intake and female infertility: a study based on NHANES database

Abstract Background This study was designed to investigate the association between nutrients and female infertility. Methods A cross-sectional study on 18–45 years of age reproductive-age women was conducted using the data from the National Health and Nutrition Examination Surveys (NHANES) for the periods 2013–2014 and 2015–2016. Multivariate logistic regression analysis was performed to evaluate the association between nutrients and female infertility. Subgroup analysis was applied to the body mass index (BMI). Results were summarised using an odds ratio (OR) with a 95% confidence interval (CI). Results Of the total 1713 women, 204 women (11.91%) were infertile. The result demonstrated that higher intake of carbohydrate (OR: 0.46, 95% CI: 0.24–0.86, p = 0.018), vitamin A (OR: 0.44, 95% CI: 0.24–0.80, p = 0.009), vitamin C (OR: 0.48, 95% CI: 0.26–0.88, p = 0.020), magnesium (OR: 0.36, 95% CI: 0.17–0.76, p = 0.009), iron (OR: 0.43, 95% CI: 0.23–0.82, p = 0.012), lycopene (OR: 0.55, 95% CI: 0.33–0.91, p = 0.022), and total folate (OR: 0.38, 95% CI: 0.20–0.70, p = 0.003) were associated with a lower risk of female infertility. The subgroup analysis also reported that intakes of vitamin A, vitamin C, and lycopene were related to a lower risk of female infertility among women with a BMI being 18.5–24.9 kg/m2. Among women with BMI > 24.9 kg/m2, high intakes of magnesium, iron and total folate were associated with a decreased risk of female infertility. Conclusions The intake of several nutrients is associated with a decreased risk of female infertility. These findings provide insight into potentially modifiable lifestyle factors associated with female infertility. Plain language summary Infertility is becoming a global challenge in both medical and social aspects. There is growing evidence of the importance of nutrition in reproduction in animal and human studies, suggesting a correlation between nutrition and female fertility. We observed that higher intakes of carbohydrates, vitamin A, vitamin C, magnesium, iron, lycopene and total folate were associated with a lower risk of female infertility. This study helped increase awareness among health professionals and patients about the important link between nutrients and infertility, and educate women about the significance of a healthy lifestyle and diet.


Introduction
Infertility is defined as the inability to conceive after 12 months of unprotected intercourse or six months for women aged 35 years or older (Vander Borght and Wyns 2018).Infertility is a global health problem affecting 20-30% of the female population of reproductive age (Silvestris et al. 2019).Infertility is more than a quality-of-life issue, with considerable public health consequences including psychological distress, social stigmatisation, economic strain, marital discord, negative pregnancy outcomes and later-onset adult diseases (Sun et al. 2019).Although male infertility contributes to more than half of all cases of global childlessness, the major psychological and social burdens primarily affect women (Westerman and Kuhnt 2022).Therefore, it is necessary to explore the factors that may affect female infertility.
A plethora of systemic and gynecological conditions can affect the female reproductive system and potentially lead to infertility, including polycystic ovarian syndrome (PCOS), endometriosis, premature ovarian failure and pelvic inflammatory disease (Deshpande andGupta 2019, Afrin et al. 2021).Besides organic diseases, lifestyle factors such as unbalanced nutrition and an unhealthy diet may interfere with the physiological reproductive functions (Afrin et al. 2021, Silvestris et al. 2019).Mounting evidence suggests a correlation between nutrition and female fertility (Silvestris et al. 2019, Aoun et al. 2021), however, conflicting results exist.In a Danish and North American preconception cohort study (Wise et al. 2018), high trans fatty acids intake and low omega-3 fatty acid intake were associated with reduced fecundity.A study (Li et al. 2019) performed in Massachusetts reported that total consumption of vitamins A, C and E before starting infertility treatment with assisted reproductive technologies was not associated with live birth rates.A review demonstrated an association between higher folate intake with increased fecundability (Gaskins and Chavarro 2018).Nevertheless, another study found that high doses of folic acid do not increase the odds of pregnancy in infertile women (Murto et al. 2014).Given the contradictory studies and there are only a few studies devoted to studying the association between nutrient intakes and female infertility, this study was conducted.
Herein, this study was to evaluate the relationship between multiple nutrients and female infertility, which may be helpful for guiding the search for appropriate dietary patterns.

Study design and participants
A cross-sectional study was conducted using data from the National Health and Nutrition Examination Survey (NHANES) database between 2013 and 2014 and 2015 to 2016 (https:// www.cdc.gov/nchs/nhanes/about_nhanes.htm).The NHANES is a program of studies conducted to understand the health and nutritional status of the United States population since the 1960s.The survey includes interviews and physical examinations in a nationwide representative sample of about 5000 participants each year.The NHANES gathers demographic, socioeconomic, dietary and health-related information.The physical examination section involves medical, dental and physiological measurements, as well as laboratory tests directed by qualified medical professionals (Anon 2020).Data from women of 18-45 years of age who were sexually experienced were analysed.Women who did not answer the question 'history of infertility', had a history of ovariectomy and hysterectomy, with missing data on total polyunsaturated fatty acids (PUFA), with missing data on sex hormone binding globulin (SHBG), and with missing data on ever use female hormones were excluded from the study.Pregnant women were also excluded.The flow chart of data selection is shown in Figure 1.As the data sets included in the study were downloaded from public databases, the study did not need the approval of an ethics committee from our hospital.We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement according to the Enhancing the Quality and Transparency of Health Research (EQUATOR) guidelines (von Elm et al. 2008).

Covariate
Covariate variables included energy (kcal), age (years), ethnicity (Mexican American, other Hispanic, non-Hispanic white, non-Hispanic black or other ethnicities), marital status (divorced, living with a partner, married, never married, separated, and widowed), the education level [9-11th grade, college graduate, high school graduate, less than 9th grade, some college or Associate of Arts (AA) degree], annual family income (less than $20,000 or $20,000 or more), body mass index (BMI, kg/m2 ), smoking at least 100 cigarettes in life (yes or no), had at least 12 alcohol drinks/1 year (yes or no), age when first menstrual period occurred (years), ever treated for a pelvic infection (yes or no), ever taken birth control pills (yes or no), ever use female hormones (yes or no), oestradiol (pg/mL), testosterone (ng/dL) and SHBG (nmol/L).

Self-reported infertility
Infertility is defined as the absence of pregnancy with unprotected intercourse for one year (Anon 2015).The presence of infertility was assessed using a self-reported questionnaire.

Statistical analysis
The measurement data of the normal distribution were described with the mean ± standard error [mean (S.E.)], and a t-test was used for comparison between groups.Counting data were expressed as numbers and percentages were compared between the two groups using the chi-square test.The missing values were filled by multiple interpolations.The missing values before and after interpolation were compared by performing a sensitivity analysis.Multivariate logistic regression analysis was used to evaluate the association between nutrients and female infertility.Model 1 was created by not adjusting for variates, and Model 2 adjusted for age, BMI, marital status, pelvic infection, SHBG and energy.The association between recommended intakes of nutrients and female infertility was also performed.The subgroup analysis was performed based on BMI.Statistical significance was defined as a p value of < 0.05.The data were analysed using SAS version 9.4 (SAS Institute, Cary, NC).

Basic characteristics of included participants
In total, 1713 women were included, comparing 204 women being infertile.The mean age of included women was 32.28 (0.26) years.The majority of the participants (46.16%) included were married.The BMI of 1114 (62.38%) participants was greater than 24.9 kg/m 2 .The basic characteristics of the included participants are presented in Table 1.The mean estimated daily intake of carbohydrates was 233.51 (3.40) g.Participants without female infertility had a higher mean estimated daily intake of vitamin A [590.63 (21.78) mcg] compared with participants with female infertility [543.12 (48.53) mcg].Participants without female infertility had a higher mean estimated daily intakes of protein, Vitamin E, Vitamin C, Vitamin D, selenium, zinc, magnesium, iron, lycopene, total folate, Beta-carotene and lutein þ zeaxanthin.Characteristics of nutrients intakes in participants with and without female infertility are shown in Table 2.There were statistically significant differences in age, marital status, BMI, pelvic infection, SHBG, total fat, vitamin C, zinc and total folate between infertile and non-infertile women (all p < 0.05).

Discussion
Infertility is becoming a global challenge in both medical and social aspects (Inhorn and Patrizio 2015).There is growing evidence of the importance of nutrition in reproduction in animal and human studies, suggesting a correlation between nutrition and female fertility (Bosdou et al. 2019, Vitagliano et al. 2021).A review assessed the evidence for the effectiveness of different antioxidants in female subfertility.However, in this review, there was low-to very low-quality evidence to show that there was low-to very low-quality evidence to show that taking an antioxidant may benefit subfertile women (Showell et al. 2020).The association of various types of nutrients with female infertility still needs evaluation.This study was conducted to evaluate the association between multiple nutrients and female infertility and indicated that higher intakes of carbohydrates, vitamin A, vitamin C, magnesium, iron, lycopene and total folate were associated with a lower risk of female infertility.In addition, intakes of vitamin A, vitamin C, and lycopene were related to a lower risk of female infertility among women with a BMI being 18.5-24.9kg/m 2 .Among women with BMI > 24.9 kg/m 2 , high intakes of magnesium, iron and total folate were associated with a reduced risk of female infertility.
The ability to sense nutrients and to adapt physiological responses to nutrient availability and characteristics allowed organisms to increase their opportunities to survive and to expand their progenies (Della Torre et al. 2014).This study did not observe the association between PUFAs and female infertility.A cross-sectional study conducted among women aged 20-44 years found that PUFA intake is only slightly associated with infertility (Wang et al. 2022).No statistical association between vitamin D and female infertility was observed in our study.However, a recent study demonstrated that vitamin D status appears to be relevant to reproductive physiology, and to physiological processes underlying common gynecological disorders as well as for reproductive success (Simpson and Pal 2023).The relationships between PUFAs/vitamin D and female infertility need further elucidation.In an Italian study of women undergoing in vitro fertilisation, the author found no association between carbohydrate consumption and in vitro fertilisation (Noli et al. 2020).However, the Nurses' Health Study II reported that a high-carbohydrate diet with a high glycemic index was associated with an increased risk of infertility due to ovulation disorders in apparently healthy women (Chavarro et al. 2009).This finding demonstrated no association between carbohydrate intake and female infertility when the carbohydrate intake was less than 287.09g.However, we found that a higher intake of carbohydrates was associated with a lower risk of female infertility.We speculate that the difference may be caused by the difference in population or intake dose.The relationship between carbohydrate intake and female infertility needs to be further elucidated through research.
Folate and folic acid are water-soluble B vitamins, also known as vitamin B9 (Scaglione and Panzavolta 2014).The two terms are often used interchangeably, but the first refers to the form naturally present in some foods, including green and leafy vegetables, sprouts, some fruits, legumes, seeds and offal, while the second is synthetic molecule introduced in supplements or fortified foods (Ferrazzi et al. 2020).In this study, total folate intake was associated with a lower risk of female infertility.Previous studies agree that the intake of folate is recommended for the prevention of neural tube defects and is related to a lower frequency of infertility (Gaskins andChavarro 2018, Czeizel et al. 2011).In a recent study, Kadir et al. reported a significant increase in the number of antral follicles in women who took folic acid daily (Kadir et al. 2022).WHO strongly recommends all women of reproductive age daily use 400 mg of folic acid, in addition to consuming food with folate from a varied diet (Gomes et al. 2016).The possible reason was that folate has been reported to be possibly capable of improving ovarian response and oocyte quality (Thaler 2014).
In this study, greater intakes of vitamin A and vitamin C were associated with decreased risk of female infertility.Vitamin A represents a group of fat-soluble retinoids including retinol and retinoic acid, which can be obtained from vegetables, dairy products, eggs and fruits (Olza et al. 2017).A previous study (Baleato et al. 2005) reflects a direct requirement for retinoids by germ cells for the resumption of spermatogenesis in vitamin A deficient animals via mechanisms that involve the suppression of BMP4 expression.Vitamin C could notably compensate undesirable effects of ovarian ageing in a mouse model (Abdollahifar et al. 2019).The evaluation of the effects of vitamin A and vitamin C on female infertility needs more studies.
In this study, a higher intake of lycopene seemed to decrease female infertility.Antioxidation may be the mechanism for the correlation between lycopene and decreased risk of female infertility.Lycopene is rich in antioxidants, more consumption of which was reported to be associated with easier conception in a shorter time in patients with unexplained infertility (Ruder et al. 2014).Reactive oxygen species are known as an important cause of female infertility and abundant antioxidants from the lycopene pattern may alleviate the oxidative damage to fertility (Agarwal et al. 2012).
The study found that higher intakes of magnesium and iron were associated with a lower risk of female infertility.Magnesium is one of the cations found in the human body that takes part in energy transformations and determines the proper course of hormonal reactions and insulin secretion (Pokorska-Niewiada et al. 2021).Mhaibes et al. observed significantly lower concentrations of magnesium in the serum of PCOS patients compared with the control group (Mhaibes et al. 2017).As the most abundant trace element in the human body, iron plays a key role in the synthesis of DNA and proteins, the production of erythrocytes, electron transport, cellular respiration, cell proliferation and the regulation of gene expression.This study demonstrated an association between the intake of iron and female infertility.High consumption of iron exacerbated hyperlipidaemia and hyperglycaemia as well as induced fatty liver changes and sterility along with a reduction of female fertility (Kim et al. 2017).Chavarro et al., analysing the relationship between the consumption of various forms of iron and ovulatory infertility, found that the use of iron supplements with high iron content was associated with a reduced risk of ovulatory infertility (G€ unalan et al. 2018).The optimal dose for magnesium and iron intake needs to be further clarified.
Based on our subgroup analyses, high intakes of magnesium, iron, and total folate were associated with a reduced risk of female infertility among women with BMI > 24.9 kg/ m 2 .The possible reason for this finding is that obese and overweight people themselves are deficient in magnesium, iron, and total folate.Magnesium deficiency is frequent in obese patients (Piuri et al. 2021).Magnesium deficiency is often associated with negative health outcomes, including low-grade inflammation (Pelczynska et al. 2022).Furthermore, low-grade chronic inflammation is often associated with several gynaecologic disorders associated with female infertility (Di Renzo et al. 2022).Similarly, one of the pathologic conditions observed in obesity is systemic iron deficiency and  hypoferremia (Nikonorov et al. 2015).A case-control study reported that ferritin levels <30 mg/L were associated with unexplained infertility (Holzer et al. 2023).Overweight and obese individuals have lower serum folate concentrations when compared with individuals with normal weight (Kose et al. 2020).Intake of supplemental folic acid, particularly at doses higher than those recommended for the prevention of neural tube defects, has been consistently related to a lower frequency of infertility (Gaskins and Chavarro 2018).
This study helped increase awareness among health professionals and patients about the important link between nutrients and infertility, and educate women about the significance of a healthy lifestyle and diet.During the daily work of physicians in the department of reproductive medicine, counselling about dietary suggestions is usually required.Testing nutritional status prior to pregnancy may help to highlight micronutrient deficiencies and plan an appropriate nutrient-rich diet.However, several limitations should not be ignored before fully understanding this article.First, because this study is based on a cross-sectional design, it is impossible to establish causal relationships among the findings.Second, important confounding factors are not available in the database, such as the frequency of sexual intercourse, and length of the menstrual cycle, which may affect our results.Third, 'infertility' is a participant's reported answer, not an explicit diagnosis of infertility, which may affect the results.More information regarding the role of nutrients in female infertility is needed to provide guidelines devoted to the nutritional management of infertile women.

Conclusions
Higher intakes of carbohydrates, vitamin A, vitamin C, magnesium, iron, lycopene and total folate were associated with a lower risk of female infertility.These findings provide insight into potentially modifiable lifestyle factors associated with female infertility.

Figure 1 .
Figure 1.Flow chart of data selection.NHANES: National Health and Nutrition Examination Surveys; PUFA: polyunsaturated fatty acid; SHBG: sex hormone binding globulin.

Figure 2 .
Figure 2. Subgroup analysis of the association between nutrients and female infertility based on BMI categories.BMI: body mass index; OR: odds ratio; CI: confidence interval; Ref.: reference.

Table 1 .
Basic characteristics of included participants.

Table 2 .
Characteristics of nutrients intakes in participants with and without female infertility.
Notes: PUFA: polyunsaturated fatty acids; mean (S.E.): mean ± standard error; t: t-test; v 2 : chi-square All nutrients intakes concentrations were divided into four groups according to the interquartile range.

Table 3 .
Association between nutrients and female infertility.

Table 3 .
Continued.OR: odds ratio; CI: confidence interval; PUFA: polyunsaturated fatty acids All nutrients intakes concentrations were divided into four groups according to the interquartile range; Model 1 was created by not adjusting for any variable, and Model 2 adjusted for age, BMI, marital status, pelvic infection, SHBG and energy.
NUTRIENT INTAKE AND FEMALE INFERTILITY