S El-Azab, A Abdelhamid, H Shalaby, A Mehrim, A Ibrahim
aflatoxin, human female infertility, ovulatory disorder
S El-Azab, A Abdelhamid, H Shalaby, A Mehrim, A Ibrahim. Study Of Aflatoxin B1 As A Risk Factor That Impair The Reproductive Performance In Females- Egypt. The Internet Journal of Toxicology. 2008 Volume 6 Number 1.
Aflatoxins are a group of naturally occurring, highly toxic mycotoxins that contain a characteristic dihydrobisfuran moiety in their molecular structures. These fungal metabolites are produced by specific strains of
Human infertility due to female factors was thought to be the reason behind all fertility problems. Whereas experts recognize that female infertility accounts for about only 40% of all infertility cases, ovulatory disorders are one of the most common reasons why women are unable to conceive, and account for 25% of women's infertility . Causes of infertility are many, such as sexually transmitted diseases, parasitic diseases, physiological and genetic defects, and toxic agents. One of the least understood among these factors seems to be the impact of toxic agents, including mycotoxins, on the reproductive performance of human beings . Studies elsewhere have shown the presence of aflatoxins in common food items in Egypt, suggesting that exposure of members of this community through diet to aflatoxins may be high . The present study was, therefore, initiated to determine whether aflatoxins are present in the blood of infertile females in this community and to ascertain whether there is a relationship between the presence of aflatoxin and deviation from normal in human female ovulatory functions, and hormonal parameters.
Subjects and Methods
Selection of the patients
Fifty patients (infertile females), mean age (31±3) years with an ovulation problems referred from the Infertility Unit, Mansoura University Hospital, were enrolled in the study. The underlying ovulation pathology caused by polycystic ovary syndrome (POCS) (n=43), and idiopathic anovulation (n=7). The exclusion criteria were patients with acute infections, and work place pollution. Twenty healthy subjects (fertile female), mean age (30± 3) were chosen as control. All the participants did not receive any supplements that could increase ovulation function within the previous six months. Informed consent was obtained from all participants.
Sampling for AFB determination
Five ml blood sample was drawn from each participant, all samples were kept frozen until analysis. The samples were tested for the presence of AFB1. The quantitative determination of AFB1 by Thin Layer Chromatography (TLC) was carried out according to the method of Eppley , modified by Abdelhamid . The laboratory studies were undertaken in the mycotoxin laboratory of Prof. Dr. Abdelhamid, A. M., Faculty of Agriculture, Mansoura University. All chemicals and solutions used were from United Co. for Chemical and Medical Preparations. Mycotoxin standard used was from Makor Chemicals Co.
5ml blood sample was extracted with 24 ml chloroform and 1ml n phosphoric acid, the chloroform layer was received and transferred into a flask and dried under vacuum on Rota vapor-M (Bűchi-HB-140) at 60°C. Residues were dissolved in 20ml chloroform for TLC spotting. Development of the plate was done in a closed developing tank for about 40 minute, with solution: Toluene/ acetic acid/ Formic acid 6:3:1. The plate was dried in air and examined under ultraviolet (U.V) at wave length 366. The verification was done through Rf value and the fluorescence colour (blue fluorescence) under U.V at 366 nm, after comparison with external standard . Confirmatory test was done when the plate was sprayed with 30% methanolic sulphuric acid (30 ml H2 so 4 + 70 ml CH3 OH) and examined again under the U.V. The blue fluorescence of aflatoxin converted to golden yellow colour.
Examination of the ovary
The ovaries for all participants were examined by measuring the mean ovarian volume; and number and size of ovarian follicles. Blood hormonal levels were determined calorimetrically.
Statistical analysis was done by using SPSS Software version 10.0 (SPSS, Chicago, IL, U.S.A.). The data were expressed as mean ± standard deviation for patients and control separately. Differences in means were analyzed using student t-test for comparison between two groups. The P- value was considered significant if less than 0.05.
Table (1) shows that all the blood samples (the control and patients' samples) were negative as regard to AFB1 analysis.
Table (2) shows that there was a significant enlargement in the mean ovarian volume in infertile females compared to controls, whilst, there was a significant increase in the mean follicular size in controls compared to infertile females group (using Trans-Vaginal Scanning).
Table (3) shows that there were a significant higher levels of LH and a significant lower levels of mid luteal progesterone in infertile females group compared to controls.
Toxicity of dietary aflatoxin in mammals has been extensively studied and proved to be inducing tissue toxicity . The present study was initiated to determine whether aflatoxin is present in the blood of infertile females in Egypt and its correlation with women ovarian functions. All the blood samples (the control and patients samples) showed negative results as regard to AFB1 analysis (Table 1). In spite of these negative results, it does not exclude the role of aflatoxin as a contributing factor may causing female infertility, as the cumulative effect of feeding low levels of mycotoxins (outspreading under the local conditions) may contribute to a gradual deterioration of organ functions, this in turn may affect fertility and overall health . This negative result may be an indicator of a chronic aflatoxicosis, through which the toxin could be deposited in its target organ (in the case of AFB1 is the liver) and not in the blood pool as in the acute toxicoses. In addition, mycotoxins (including aflatoxins) negatively affect reproductive system of various animal species , since mycotoxins-contaminated diets led to some significant changes in egg characteristics and composition . Moreover, aflatoxin is found normally in humans more frequently in females than in males (although males are more sensitive for aflatoxin than females) .
Furthermore, there are many reports about the deleterious effects of aflatoxin on the reproduction system, i.e. sexual maturation, growth and maturation of the follicles, levels of hormones, gestation, and growth of foetus . Therefore, aflatoxin lowered the fertility to 13 % and increased the mortality of embryos . However, Kihara
Anovulation is the cause of infertility in about a third of couples, polycystic ovary syndrome accounts for 90% of such cases . Polycystic ovary syndrome can be confirmed by the presence of two of the following criteria: biochemical or clinical hyperandrogenism, menstrual irregularity, and polycystic ovaries on ultrasound . Environmental toxins may play a role in the pathogenesis of anovulatory infertility especially polycystic ovary syndrome . In the present results, there was a significant enlargement in the mean ovarian volume in infertile females compared to controls (common criteria in POCS); whilst there was a significant increase in the mean follicular size in controls compared to infertile females group (Table2). These findings may be referred in part to the toxic effects of aflatoxin as proved by Abd El–Wahhab , who reported that, microscopic examination of the ovaries of female rabbits treated with 0.15mg AF B1/kg BW showed some pathological alterations in the form of coagulative necrosis which appeared mainly in the growing and mature follicles, and decrease in number and size of Graffian and growing follicles with increased number of atretic follicles and small areas of degenerative changes.
The pronounced high levels of LH concentration in infertile females compared with controls may be attributed to a direct effect of increasing its basal level from anterior pituitary and / or secretion of Gonadotrophic Releasing Hormone (GnRH) from hypothalamus. Also, such increase might be associated with increasing oestradiol to maximum level. On the other hand, the marked increase in progesterone level during mid luteal phase in controls reflected normal Corpus Luteum (CL) formation. While, the opposite which was observed in infertile females (i.e. the lower levels of progesterone) is due to direct effect of reduced CL size, which was indicated from the significant higher levels of LH in infertile females compared to controls. Two unrelated toxic actions have been suggested to explain the AFB1 effects on female's fertility: An indirect effect mediated by AFB1 induced hypovitaminosis A; and a direct antagonistic interaction with steroid hormones receptors interfering with gonadal hormones production as estrogen and progesterone, due to structural similarity of AFB1 and steroid hormones . This results support our results as there was a significant lower levels of mid luteal progesterone, with higher ovarian volume in infertile females group compared to controls. This could be explained by interfering with the production of progesterone from the un-ruptured ovarian follicles. Furthermore, AFB1 negatively affects hepatic alphafetoprotein (AFP) synthesis, and AFP has shown to cause genital function blockade which leads to reduced levels of hormonal promoters .
It has been proven that, fertility of pregnant rats decreased after aflatoxin and embryonary resorptions, malformations and developmental retardations occurred . Moreover, chronic exposure to aflatoxin decreased reproduction efficiency of ruminants . A deleterious effect on the gonads and embryo-toxicity in the experimental animals were observed. There were increases in fetal resorption, implantation loss, and intra-uterine death. The data showed disturbances in oestrus cycle, significant reductions in the number of oocytes and large follicles, inhibition, and reduction in the conception rates . It can be