A Comparative Study of Vitamin D Serum Levels in Monosymptomatic Enuretic Children and Non-enuretic Children
Conditions: Nocturnal Enuresis
Sponsor: Ain Shams University

A comparative study of Vitamin D serum levels in monosymptomatic 
enuretic children and non-enuretic children 
INTRODUCTION 
Nocturnal enuresis is a widely prevalent problem. It occurs in up to 15% of 5-year-old 
children and 10% of 10-year-old, with a spontaneous remission rate of 14% per year. 
Bedwetting can have a considerably distressing social impact on children and subsequently 
their families, affecting children’s self-esteem and interpersonal relationships, and their 
scholastic achievement. () 
Nocturnal enuresis is classified into monosymptomatic (MNE) and non-
monosymptomatic (NMNE), depending on whether it is associated with voiding or storage 
symptoms or not. It can also be categorized into primary, which is since birth, and secondary 
enuresis, which is newly onset after at least 6 months of a previous asymptomatic state. (2) 
Primary monosymptomatic nocturnal enuresis (PMNE) is the most frequent urologic 
complaint among pediatric patients; however, the pathology and etiology of this problem have 
not been clearly determined. (3) 
Nocturnal bladder control is a matter of developmental delay. At 1 year of age, the 
conscious sensation of urinary bladder fullness starts to evolve and this physiological turning 
point allows the beginning of the development of voluntary control of voiding by the age of 2 
years. (4) By the age of 4 years, full daytime and nighttime urinary control is obtained by most 
children. (5) 
The idea that enuresis is related to the immaturity of the central nervous system is 
supported by the finding that premature and/or low-birth-weight children experienced bed-
wetting more often than normal-birth-weight children. (6) 
Low nocturnal anti diuretic hormone (ADH) and urine osmolality play a role in MNE 
associated with Nocturnal Polyuria (NP), which doesn’t occur as an isolated abnormality but 
is also linked to children who also suffer of arousal abnormality (7). We have proved this in our 
previous study in which we stated that Nocturnal polyuria and decreased nocturnal urine 
osmolality were key factors in pathogenesis of nocturnal enuresis (7).  
There are many clinical observations that link nocturnal enuresis with developmental 
delay in language and physical growth (8) and skeletal maturation. (9) Nocturnal enuresis could 
be linked with sleep disorders as well. (10) 
Vitamin D is important for bone growth and regulation of calcium homeostasis. It not 
only plays a vital role in skeletal growth but also has other critical biological actions in neural 
development and function. (11) 
The source of vitamin D is primarily sunlight; it is also found in few foods as oily fish 
and fortified margarine. Vitamin D consists of a cholesterol backbone and has steroid-like 
effects. It is the fat-soluble hormone that plays an essential role in brain health. (12) 
Studies claim that vitamin D may have an essential role in sleep regulation (13). 
Vitamin D deficiency is linked with Obstructive Sleep Apnea (OSA) (14) and nocturnal 
polyuria is one of the main adverse outcomes of OSA. (15) 
Vitamin D deficiency could cause excessive urine production directly. This was shown 
in a study done on mice which showed that vitamin D deficiency leads to activation of the 
Renin Angiotensin system. Eventually the augmentation of renin synthesis leads to increased 

plasma Ang II production from angiotensinogen, which drives mice to increase water intake 
and intestinal salt absorption, since Ang II is a very potent thirst-inducing agent that acts on 
the central nervous system, as well as a potent stimulator of intestinal sodium absorption. As a 
result, the mice must excrete more urine and salt to maintain volume and electrolyte 
homeostasis. (16) 
AIM OF THE STUDY 
To evaluate the relationship between Vitamin D deficiency and nocturnal enuresis, and 
to study the correlation between vitamin D deficiency and urine osmolality which is an indirect 
indicator of ADH deficiency. 
PATIENTS AND METHODS 
This is a case control study which was performed in Ain Shams University Hospitals. 
It was conducted in the period between February 2021 and August 2021. 
Patient group: Sixty children aged from 5 to 15 with primary mono-symptomatic nocturnal 
enuresis were included and selected randomly.  
Inclusion criteria:  Patients with PMNE which was defined as bedwetting frequency of 3 or 
more nights per week for at least 2 weeks of observation, and who didn’t receive any previous 
treatment for enuresis. 
Exclusion criteria: Patients with non-monosymptomatic enuresis (associated with storage 
and/or voiding symptoms), anatomical abnormalities (Meatal stenosis, Hypospadias, 
Epispadias, etc.), neurological diseases, renal impairment, previous pelvic, urological or brain 
and spinal cord surgery, chronic constipation and irritable bowel habits, active urinary tract 
infection and patients who are on vitamin D supplements. 
Control group: Sixty healthy children were included as a control group whose ages ranged 
from 5 to 15 years old, and they were chosen from apparently healthy non-enuretic children. 
Written informed consent was obtained from patients’ legal guardians, and the study 
was approved by Ain Shams University Ethical Committee. 
Methods:  
All children included in the study were subject for detailed medical and surgical history to rule 
out the presence of organic or psychological diseases. Physical examination including focused 
neurological examination to exclude neurological diseases, especially (neural tube defects) and 
local urological examination to check for any genital or external urethral meatus anomalies. 
Also urine analysis was done to exclude urinary tract infection. 
Frequency Volume chart and nocturnal urine osmolality. 
Volume Frequency chart was recorded for both enuretic and control groups, to evaluate 
frequency, average voided volume, and to exclude or confirm the presence of nocturnal 
polyuria.  
Nocturnal polyuria is defined in children and adolescent age group as, urine production during 
the period of sleep which equals to or more than 130% of estimated bladder volume for age 
(EBV). (17) 
Estimated bladder capacity for age is calculated by the formula [30 X (age in years + 2)] in ml. 
The amount of urine last voided before sleep is discarded and first morning voiding volume is 

added to the calculated nocturnal urine volume. Clear instructions for mothers were to let the 
child void before sleep and every two hours till 8 am. Short intervals were meant to avoid 
enuretic episodes during calculation of nocturnal urine volume, and if occurred it mandates the 
repeat of chart on another day. 
Nocturnal urine collected over the night was tested for osmolality, it was measured in 
mOsm/kg. 
Serum Vitamin D level.  
Vitamin D serum level was measures using the ELISA technique. The Cal Biotech Vitamin D Kit 
(Mannheim, Germany) was used, which is a solid phase enzyme linked immunoassay (ELISA), 
based on the principal of competitive binding. Vitamin D deficiency was defined as serum Vit. 
D below 20 ng/ml. 
Sensitivity:  
The sensitivity of this test kit is 0.67ng/ml. Sensitivity was determined by calculating 
the mean plus 2SD of the standard zero point tested 20 times in the same Statistical Methods 
run. 
Statistical methods 
The collected data were coded, tabulated, and statistically analyzed using IBM SPSS statistics 
(Statistical Package for Social Sciences) software version 28.0, IBM Corp., Chicago, USA, 
2021. Quantitative data tested for normality using Shapiro-Wilk test, then described as 
mean±SD (standard deviation), and then compared using independent t-test. Qualitative data 
described as number and percentage and compared using Chi square test. Pearson test was 
used for correlations. ROC curve was used to evaluate the performance of variables to 
differentiate between conditions. The level of significance was taken at p-value <0.050 was 
significant, otherwise was non-significant.   
Diagnostic characteristics was calculated as follows: 
- 
Sensitivity = (True positive test / Total positive golden) x 100 
- 
Specificity = (True negative test / Total negative golden) x 100 
- 
Diagnostic accuracy = ([True positive test + True negative test] / Total cases) x 100 
- 
Youden's index = sensitivity + specificity – 1 
- 
Predictive positive value = (True positive test / Total positive test) x 100 
- 
Predictive negative value = (True negative test / Total negative test) x 100 
- 
Likelihood ratio positive (LR+) = (sensitivity/ 1-specificity)  
- 
Likelihood ratio negative (LR-) = (1- sensitivity / specificity) 
Diagnostic odds ratio (DOR)= LR+/ LR- 
 
 
 
 
 
 
 
 
 

Serum level of vitamin D, in monosymptomatic enuretic children and 
non-enuretic children: a comparative study 
Dr. Diaa eldin Mostafa*, Prof. Dr. Hasan Shaker*, Dr Abdallah Badr**, Dr Mohamed 
Abuelnaga*.  
*   Urology Department, Faculty of Medicine, Ain Shams University 
** Damanhour National Medical Institute. 
 
Corresponding Author: 
Dr Diaa Eldin Mostafa  
Associate professor of urology 
Faculty of Medicine 
Ain shams university 
 
Email: Diaamahmoud@yahoo.com 
Mobile No: +201228044081 
Egypt, Cairo, El Abbassia square, Faculty of medicine, El Demerdash hospital. 
 
Dr Hassan Shaker 
hassanshaker@live.com 
Professor and chief of urology department 
Faculty of medicine 
Ain Shams university 
 
Dr Mohamed Abulnaga  
Abuelnagam@yahoo.co.uk 
Lecturer of urology 
Faculty of medicine 
Ain shams university 
 
Dr Abdallah Badr 
torbido14@gmail.com 
Lecturer of urology 
Damanhour National medical institute 
 
 
 
Word count: 2407 
 
Ethical committee approval reference number: FMASU MS 152/2020 
No funding sources to be disclosed research was conducted in Ain Shams university 
Hospital. 
No conflict of interest to be disclosed. 
Informed consent was obtained from all patients or their legal gaurdians before 
recruitment in the study. 
We herby approve any copy rights for publication by the Journal. 

Cover letter 
 
 
Dear editorial Board: 
We are group of researchers interested in causes and management of nocturnal 
enuresis, we embarked on this research last year and we reached promising results regarding 
the role of vitamin D in management of monosymptomatic enuresis. 
This research was conducted in Ain Shams university hospitals after approval of our 
university ethical committee, no external funds whatsoever was received for this study, and 
no conflict of interest to be declared. 
This study was continuation of our previously published study about the etiology of 
monosymptomatic nocturnal enuresis. 
Title was “Nocturnal polyuria and nocturnal arginine vasopressin (AVP): A key factor in the 
pathophysiology of monosymptomatic nocturnal enuresis. 
Neurourology and Urodynamics  
2009 | Journal article  
DOI: 10.1002/nau.20697  
 
Thanks, and much obliged.  
 Corresponding author  
Diaa Abdelfattah Mostafa MD