Novel cord blood and urinary phytoestrogens levels in male neonates with normal external genitalia

Che Che Ahmad Muttaqin Salim; Azrina Shahrizat Khutubul Zaman; Hana Azhari; Suzana Makpol; Shareena Ishak; Zarina Abdul Latiff; Dayang Anita Abdul Aziz

doi:10.4103/jcn.jcn_95_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 12 | Issue : 1 | Page : 16-21

Novel cord blood and urinary phytoestrogens levels in male neonates with normal external genitalia

Che Che Ahmad Muttaqin Salim¹, Azrina Shahrizat Khutubul Zaman¹, Hana Azhari², Suzana Makpol³, Shareena Ishak⁴, Zarina Abdul Latiff⁴, Dayang Anita Abdul Aziz¹
¹ Department of Surgery, Pediatric Surgery and Urology Unit, Faculty of Medicine, National University of Malaysia, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
² Department of Family Medicine, National University of Malaysia, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
³ Department of Biochemistry, National University of Malaysia, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
⁴ Department of Pediatrics, Faculty of Medicine, National University of Malaysia, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

Date of Submission	12-Oct-2022
Date of Decision	14-Nov-2022
Date of Acceptance	15-Nov-2022
Date of Web Publication	03-Jan-2023

Correspondence Address:
Dayang Anita Abdul Aziz
Department of Surgery, Pediatric Surgery and Urology Unit, Faculty of Medicine, National University of Malaysia, Universiti Kebangsaan Malaysia, Kuala Lumpur 56000
Malaysia

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jcn.jcn_95_22

Abstract

Objective: This was a new prospective study to determine the association between phytoestrogen levels in the cord blood and urine with normal and abnormal external genitalia in male newborns. Materials and Methods: One hundred and fifty-one term male newborns were enrolled. Cord blood and urine and medical photos of their external genitalia were obtained. Prematurity and multiple congenital anomalies or syndromic characteristics were excluded. Serum and urine levels of phytoestrogens (daidzein and genistein) were calculated using liquid chromatography/mass spectrometry. The normality of the appearance of the external genitalia of the males was determined by a consensus review by experts. Results: A total of 146 cord blood and 96 newborn urine samples were obtained; only two newborns had abnormal external genitalia. In the 144 babies with normal external genitalia, mean levels were determined to be as follows; serum daidzein 10.82 ng/ml, serum genistein 42.17 ng/ml, urine daidzein 69.91 ng/ml, and urine genistein 126.09 ng/ml. In the two babies with abnormal external genitalia, mean levels of each of the measured phytoestrogen were lower; however, there was no statistical significance between the two groups (P > 0.05). Conclusions: We successfully determined and developed a novel database on the levels of measurable phytoestrogens in serum and urine from male newborns with normal external genitalia. In the normal group, a higher concentration of serum and urinary phytoestrogens was correlated with the extension of the scrotal pigmentation above and proximal to the base of the penis. These data are useful to better understand the role of phytoestrogens in the development of male genitalia and for future research on newborns with abnormal external genitalia.

Keywords: Cord blood, male genitalia, newborn, normal, phytoestrogens, urine

How to cite this article:
Salim CC, Zaman AS, Azhari H, Makpol S, Ishak S, Latiff ZA, Abdul Aziz DA. Novel cord blood and urinary phytoestrogens levels in male neonates with normal external genitalia. J Clin Neonatol 2023;12:16-21

How to cite this URL:
Salim CC, Zaman AS, Azhari H, Makpol S, Ishak S, Latiff ZA, Abdul Aziz DA. Novel cord blood and urinary phytoestrogens levels in male neonates with normal external genitalia. J Clin Neonatol [serial online] 2023 [cited 2023 Mar 21];12:16-21. Available from: https://www.jcnonweb.com/text.asp?2023/12/1/16/366896

Introduction

Phytoestrogens are a diverse group of natural plant-derived compounds that can mimic estrogen due to their structural similarity to estradiol (17-β-estradiol). Therefore, they are capable of binding to estrogen receptors, both estrogen alpha and estrogen beta, and thus act as both an estrogenic (agonist) and anti-estrogenic (antagonist) agent. Phytoestrogens can be broadly divided into three main groups, which are isoflavones, coumestans, and lignans.

Phytoestrogens are not generated within the human body system but are consumed by eating phytoestrogenic plants and dietary estrogenic foodstuffs (soybeans, beansprouts, vegetables, grains, and drinks). Babies receive phytoestrogens through the maternal diet during pregnancy as phytoestrogens can cross the placental barrier and through breastfeeding or soy-based formula feed during infancy. External male genitalia comprises the penis, scrotum (with or without testis), and urethral opening. Several animal studies established that the estrogen receptor-alpha is the main contributor to estrogen-mediated fetal testis dysgenesis and undescended testis. In an epidemiological study among boys in Serbia, the incidence of abnormal external genitalia was 27.8%, and the causative factors were unknown.^{[1],[2],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12]}

It is nearly impossible to limit the intake of phytoestrogens in our local context of Asia. To date, there has been no study to measure and correlate the level of phytoestrogens in male babies and the pathology of their external genitalia (micropenis, hypospadias, and penoscrotal transposition); descendent or undescended testes. Therefore, this is a novel study investigating the association between the level of phytoestrogens in male newborns with the normality and pathology of the external male genitalia.

Materials and Methods

This was a prospective study conducted at the Universiti Kebangsaan Malaysia Medical Center (UKMMC), Faculty of Medicine UKM over 2 years. UKMMC is a tertiary teaching hospital for UKM, one of the renowned research universities in Southeast Asia. Universal sampling was performed for all term male newborns delivered to UKMMC who met the following inclusion and exclusion criteria. Inclusion criteria were term male newborns delivered at the end of 37 weeks of gestation, a singleton, or twins, regardless of the mode of delivery. The excluded criteria were parents with noncitizenship, parents who did not give consent to this study, patients with undescended testis but could not come to an appointment at 6 months of age, and newborns delivered with multiple congenital abnormalities or syndromic characteristics.

Pregnant mothers with an expectant male fetus from antenatal scan findings were identified for this study during the antenatal clinic in the third trimester or upon admission to the antenatal ward of UKMMC. Consent was taken with the emphasis that the baby was recruited if delivered at term with gross features of the male external genitalia and met the criteria mentioned above. Upon delivery, at least 12 ml of cord blood sample was collected in heparinized tubes. A minimum of 10 ml of neonate urine was collected later within 24–48 h of life in normal urine containers. These samples were analyzed later using liquid chromatography and mass spectrometry detection to investigate the occurrence of isoflavone aglycones (genistein and daidzein), isoflavone glycosides (genistin and daidzin), and coumestans (coumestrol) in the cord blood and urine. Analyses were conducted using a Q Exactive Orbitrap HF mass spectrometer (Fisher Scientific, Malaysia). Data analysis was carried out using Thermo TraceFinder General LC. All values are expressed as ng/ml. The inter and intraday precisions of the method were determined using six replicates of human plasma samples from quality control. The extraction and chromatographic procedures enabled the measurement of phytoestrogens in plasma, as well as in urine. Meanwhile, after delivery, the team examined the external genitalia, checking the size of the gross penis, the presence or absence of penoscrotal transposition, the presence or absence of hypospadias, and the presence or absence of undescended testes. At the same time, the external genitalia were photographed in an anterior and a lateral view and subsequently reviewed by two senior experts who would reach a consensus decision on the normality or abnormality of the external genitalia.

Statistical analysis

All statistical analyzes were performed with Statistical Package for Social Sciences® version 22. The correlation between phytoestrogen concentrations within each sample and the paired samples was analyzed using Spearman's correlation. A P < 0.05 was considered statistically significant.

Results

Study subjects' information

A total of 287 expectant mothers were recruited, but only 151 male newborns [shown in [Table 1]] were enrolled in this study as their genitalia were evaluated and photographed. Most of the enrollments were of Malay ethnicity (n = 131, 86.8%).

Table 1: Newborns and ethnicity

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External genitalia findings

The majority had a normal appearance of the external genitalia appearance (n = 149; 98.7%) [shown in [Figure 1]a and [Figure 1]c]. Abnormal external genitalia were only found in two subjects, of which one had hypospadias with chordee and bifid scrotum [shown in [Figure 1]b], whereas the other had glandular hypospadias.

Figure 1: Examples of normal, abnormal, and variant of normal external genitalia in male babies. (a) Normal genitalia: the scrotal hyperpigmentation (dotted white line) is below and distal to the base of the penis, the urethral opening is normal, there is no chordee and both testes have descended. (b) Abnormal external genitalia: hypospadias with severe chordee and a bifid scrotum. (c) "Variant" of normal external genitalia: the scrotal hyperpigmentation is above and proximal to the base of the penis, whereas the other features are normal

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Serum and urine specimen collection

Of the 151 subjects enrolled and photographed in their external genitalia, only 146 sera were obtained and 94 urine samples were collected. This means that from 149 subjects with normal external genitalia, only 144 sera and 94 urine specimens were obtained for laboratory analysis. The two abnormal external genitalia had serum and urine samples. A total of 94 subjects (92 with normal genitalia and two with abnormal genitalia) had complete paired serum and urine samples.

Initially, the serums and urine collected were analyzed for three phytoestrogens: genistein, daidzein, and coumestrol. Genistein was found to be the most detected in both types of samples, with coumestrol the least [shown in [Figure 2]]. Many factors could contribute to this low yield of coumestrol results, including the possibility of biochemical instability or an inefficient laboratory technique; hence, the decision was made to omit the coumestrol analysis as it was too costly. [Figure 3] summarized the overall numbers of samples analyzed for daidzein and genistein accordingly.

Figure 2: Detected results of phytoestrogens in all specimens

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Figure 3: Summary of the number of subjects and specimens

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Serum and urinary phytoestrogens in normal genitalia

Of the 144 serum samples of subjects with normal genitalia analyzed, 126 (87.5%) and 138 (95.8%) were detected for daidzein and genistein, respectively. While for 94 urine specimens of normal genitalia, 81 (86.2%) and 84 (89.4%) were detected for daidzein and genistein accordingly. Ninety-two newborns with normal external genitalia had complete paired serum and urine samples. The following [Table 2] summarized the quantitative results, containing mean, median, standard deviation, 95% confidence interval around the mean, and ranges for each group.

Table 2: Phytoestrogen concentrations in serum and urinary specimens of newborns with normal external genitalia

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In the cord serum sample, there was a statistically significant, strong positive correlation between daidzein and genistein, r_s (142) = 0.681, P < 0.05. Within the urine samples, there was also a statistically significant strong positive correlation between daidzein and genistein levels, r_s (142) = 0.574, P < 0.05.

It was also observed that within the highest concentrations of serum and urinary phytoestrogens, most normal external genitalia exhibit a common characteristic of having scrotal pigmentation extended above and proximal to the base of the penis, which we called "variant" [shown in [Figure 1]c]. In clinical practice, some clinicians may assume the appearance is "abnormal" and some may not be able to completely be certain that this appearance is normal; hence, the term "variant" is used. These "variant" features were also observed in a total of 84 (57%) of 149 normal external genitalia examined earlier, having varied serum and phytoestrogen readings. The correlation was, however, not statistically significant. Further implications regarding these variant external genitalia are still unknown. Functionally, the "variant" group has a normal urinary stream.

In the 92 subjects with complete paired serum and urine samples, there was a statistically significant and positive correlation between daidzein levels in serum and urine samples, r_s (90) = 0.403, P < 0.05. For genistein concentrations, there was also a statistically significant but weak correlation between these paired serum and urine samples, r_s (90) = 0.262, P < 0.05.

Serum and urinary phytoestrogens in abnormal genitalia

As there were only two abnormal external genitalia in this study, only mean levels were calculated. The mean serum daidzein was 2.76 ng/ml (range 2.52–3.00 ng/ml). The mean urine daidzein was 37.76 ng/ml (range 13.64–61.88 ng/ml). The mean serum genistein was 17.09 ng/ml (range 16.05–18.14 ng/ml). The mean genistein in the urine was 72.99 ng/ml (range 28.63–117.35 ng/ml).

Discussion

This novel study aimed to determine phytoestrogen levels in cord blood and urine samples from male newborns and to find any association between normal and abnormal external genitalia. Due to the small number of abnormal male genitalia found in this study (only two patients), no statistical significance could be demonstrated between phytoestrogen levels and abnormal external genitalia. This very small number was not surprising for a study in a single center at a teaching hospital in a city with a few other tertiary centers for maternal deliveries. Another possible factor was the strict inclusion criteria that only term newborns were enrolled, which were delivered at the end of 37 weeks of gestation.

There was also a coincidental finding of a group of patients whom we classified as having a "variant" form of external genitalia outside of the normal external genitalia group. This distinct group has similar characteristics of its scrotal pigmentation extended above and proximal to the base of the penis and was found to have a much higher concentration of serum and urinary phytoestrogens. Due to the wide range of levels demonstrated, it was impossible to calculate the range and its significance. However, this distinct group of external genitalia deserves further attention in future studies.

Serum genistein was identified the most in 95% of our serum samples and serum daidzein was identified in up to 90% of our patients. However, the serum coumestrol yield was very low, with <2% of the coumestrol level identified in the serum; therefore, the decision was made to omit this phytoestrogen from further analysis.

Urine genistein was also identified the most in almost 90% of the urine samples collected, and urine daidzein was identified in 86% of the urine samples. Similarly, urine coumestrol was the lowest identified in only 53% of urine samples analyzed; therefore, it was omitted from the analysis.

These results demonstrated that levels of daidzein and genistein were more consistent and reliable to determine should phytoestrogen levels be measured in any future studies. It was also observed that genistein concentration was consistently higher than daidzein in both serum and urine specimens, as previously reported in other studies. This again showed that genistein was more bioavailable than other phytoestrogens. However, many other variables, including maternal factors such as lifestyle and diet intake, in addition to the genetic and metabolism rates of phytoestrogens, play an important role in determining phytoestrogen levels.^{[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23]}

Genistein and daidzein have been reported to reach their maximum concentration levels in plasma about 6 h after consumption of soy food and their half-lives are about 8 and 6 h, respectively. In our study, we did not determine the time of soy food consumption by mothers before delivery. Perhaps it could have reached peak or half-life, making these phytoestrogens metabolized on the maternal side. In contrast, genistin is hydrolyzed to genistein, resulting in a continuous supply of genistein and genistin until both are excreted. Similarly, daidzein levels were suggested to be related to their metabolite formation, which has a linear relationship with the intestinal microflora.^[24]

Previous studies have proven that phytoestrogens were transferred from the mother to the fetus, crossing the placental barrier and possibly also from the amniotic fluid through the recirculation system. However, on the fetal side, it was postulated that the metabolic and excretion rate of phytoestrogen was low, therefore, the concentrations were significantly maintained.^{[11],[23],[25]}

Based on our results, the detection rate of phytoestrogens was similar between urine and serum samples; therefore, urine specimen is a feasible option for the measurement of phytoestrogens levels in future studies with less potential ethical and practical issues compared to obtaining blood samples in infants. However, in our study, urine samples were most forgotten or missed opportunities for collection within the first 48 h of life during the early phase of the study, resulting in a reduced number of samples collected.

Limitations

There were limitations in this study, mainly because our sample size was small as it only involved a single institution with a budget and time limitation of the authors to extend this study further. However, we have now been able to demonstrate that certain phytoestrogens can be measured in any serum and urine of male newborns with normal or abnormal genitalia.

Conclusions

To the best of our knowledge, similar research has not been conducted before in examining the association between phytoestrogen levels in newborns themselves and the normality of their external genitalia. Significant documented values of daidzein and genistein serum and urine in male babies with normal external genitalia serve as an important reference to understanding what would be abnormal values in future studies. To improve the results and its clinical application, we hope that a multicenter study could be carried out to truly determine the association of phytoestrogens with abnormal external genitalia in male newborns.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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