Introduction

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 Aspergillus flavus and Aspergillus parasiticus. [₁]. The most common form of aflatoxins is aflatoxin B₁ (AFB₁). Aflatoxin constitutes a real threat to the health of livestock as well as humans by their continuing intermittent occurrence in both feeds and foods [_2345678]. Several factors may enhance the occurrence of mycotoxin in the human diet in developing countries. These include eating habits, existing marketing problems which encourage long storage periods; the pre and post harvest practices that encourage build up of moisture and thus encourage mould growth, ignorance and poverty. This is aggravated by the fact that there are no strict regulations that impose limits on the concentration of mycotoxins in crops that is marketed in these countries as well as lack of relevant technology required in monitoring fungi and mycotoxins in the grains [₉]. Aflatoxin B₁ has been reported to have a deleterious effect on the reproductive capacity of laboratory and domestic female animals [₁₀₁₁₁₂]. There were reductions in ovarian and uterine sizes, increases in fetal resorption, implantation loss, and intra-uterine death in the aflatoxin exposed female rats [₁₃]. Histopathological examinations of the ovaries in aflatoxin exposed mature domestic fowls showed follicular atresia, accompanied with cessation of egg production during the whole feeding period [₁₄].

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 [₁₅]. 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 [₁₆]. 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 [₁₇₁₈₁₉]. 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

Sampling for AFB determination

Methods

Examination of the ovary

Statistical analysis

Results

Table (1) shows that all the blood samples (the control and patients' samples) were negative as regard to AFB₁ analysis.

Figure 1

Table 1: Data of AFB determination in human female blood samples under investigation (ppb).

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).

Figure 2

Table 2: The individual and ultrasound parameters of infertile females compared to controls (means ± SD).

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.

Figure 3

Table 3: Hormonal profile in infertile females compared to controls (means ± SD).

Discussion

Toxicity of dietary aflatoxin in mammals has been extensively studied and proved to be inducing tissue toxicity [₁₃]. 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 [₂₂₄]. 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 [₃₂₃], since mycotoxins-contaminated diets led to some significant changes in egg characteristics and composition [₂₄]. Moreover, aflatoxin is found normally in humans more frequently in females than in males (although males are more sensitive for aflatoxin than females) [₆₂₅].

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 [₂₆]. Therefore, aflatoxin lowered the fertility to 13 % and increased the mortality of embryos [₂₇]. However, Kihara et al. [₂₈] indicated that prenatal exposure to AFB₁ produced a delay of early response development, impaired locomotor coordination, and impaired learning ability in the offspring of rats exposed to AFB₁ during middle pregnancy, and the early gestational exposure appears to produce more effects than latter exposure. On the other hand, although, detection of AFB₁ in _{blood samples have been confirmed by TLC [6], HPLC, as well as by mass spectral analysis with standard errors [29], aflatoxin-serum albumin adduct detection may be more accurate as it has a longer half-life in animals and possibly in humans than low molecular weight aflatoxin derivatives which may be rapidly excreted. A large portion of aflatoxins found in human serum probably exist in adduct forms. Among them the AFB1-albumin adduct which is considered as a useful biomarker reflecting long- term exposure to aflatoxins in different populations [30]. The previous findings may explain why aflatoxin B1 was negative in blood samples of infertile females although it may be accumulate in organ tissues including the ovaries. On the other hand, susceptibility to aflatoxin mainly depends on its liver detoxification systems, genetic make up, age and other nutritional factors [31].}

Anovulation is the cause of infertility in about a third of couples, polycystic ovary syndrome accounts for 90% of such cases [₃₂]. 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 [₃₃]. Environmental toxins may play a role in the pathogenesis of anovulatory infertility especially polycystic ovary syndrome [₃₄]. 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 [₃₅], 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 [₃₆]. 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 [₃₇].

It has been proven that, fertility of pregnant rats decreased after aflatoxin and embryonary resorptions, malformations and developmental retardations occurred [₃₈]. Moreover, chronic exposure to aflatoxin decreased reproduction efficiency of ruminants [₃₉]. 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 [₄₀]. It can be concluded that, although the results showed negative blood samples as regard to AFB₁, it does not exclude the role of aflatoxin as a contributing factor might causing female infertility, more studies involving ovarian biopsies to analyze aflatoxin levels are recommended to study this relationship.