Environmental factors such as daylight, climate and food sources are very important for the reproduction of living things and for the offspring to continue to live in a healthy way [39, 40]. Female reproductive system is more sensitive to the negative effects of irregular, unbalanced and malnutrition conditions [40]. However, there is not a comprahensive study related to the nutritional behaviour and fertility in females. In present study, the effects of diets enriched with carbohydrate, protein, and fat were investigated on female reproductive system and infertility.

Malnutrition can affect the age of puberty [3] and cause many metabolic diseases [7,8,9,10,11]. However, no difference was observed in the pubertal age of the rats between the groups in the present study. Imbalanced intake of macronutrients may also cause serious health problems such as infertility due to estrous irregularities [41]. Mandl et al. measured the regular estrous cycle length in rats as 4.4 days [34]. Similarly, in our study, the mean estrous cycle length was found to be 4.4 days in the control group. However, a prolonged estrous cycle was detected in the protein (5.0 days) and fat groups (4.6 days). Similar to our findings, it was shown by Lilia BS et al. that 18.3% of obese women develop oligomenorrhea (prolonged estrous cycle) and 11.7% develop amenorrhea (shortened menarche phase) [42]. According to the our observations, the shortening of estrous indicates that protein and fat-based diet may cause amenorrhea, while the lengthening of diestrus indicates that protein and fat-enriched diet may cause oligomenorrhea. In the study conducted by Zheng T et al., it was shown that diets with high protein and fat content weaken the immune system [43]. Since the diestrus is the regeneration phase of the uterine endometrium, a significant prolongation of this phase, in our study, suggests that it may be related to the weakening of the immune system due to long term protein- and fat-enriched nutrition.

In a study concluded by Teleni et al., it is stated for cattle that protein diet causes an increase in blood glucose levels and increases ovulation due to the high energy associated with it [44]. However, in a study conducted on humans by Irene S et al., high protein diet caused a decrease in the number of antral follicles [45]. In other words, some studies show that a high protein diet induces folliculogenesis, and some studies show that although it induces folliculogenesis, it causes the folliculogenesis process to be incomplete. In addition, many studies with a high-fat diet have shown that a high-fat diet also causes a decrease in follicle reserve [46]. Similar to the literature, in our study, the decrease tendency in primordial follicle reserve in protein and fat groups, may be due to advanced induction of folliculogenesis. However, the decrease in the number of healthy graafian follicles and the increase in the number of atretic follicles and caspase-3 immunoreactivity indicate that the fat and protein induced folliculogenesis process cannot be completed properly. Although high protein and fat diets induce folliculogenesis, the most obvious evidence that it negatively affects the normal folliculogenesis process is the number of atretic follicles. In a study conducted by Wang Na et al., it was shown that a high-fat diet increased the number of follicles induced for folliculogenesis, but also significantly increased the number of atretic follicles compared to the control [47]. Similarly, the number of atretic follicles was found to be higher in the fat group than in the control group in our study. Furthermore, corpus luteum numbers were found to be similar between the groups, which implies that similar number of ovulation occurs in all diet groups although folliculogenesis is induced in fat and protein groups, representing the impaired folliculogenesis process.

In a study conducted by Akbariasbagh F et al. on oocytes obtained from humans, it was shown that the fertilization rate of low-volume oocytes was significantly lower than the fertilization rate of normal-volume oocytes [48]. In another study conducted on goats by Begona A et al., it was reported that healthy embryos did not form as a result of fertilization of oocytes with a diameter of less than 110 μm and excessively large (> 135 μm) oocyte volume also caused a decrease in the number of healthy developing embryos [49]. In short, if the oocyte volume is too small or large, it negatively affects fertility. In our study, for the diet groups, significant differences were observed in oocyte diameters in primary, secondary and graafian follicles compared to the control group. This suggests that long-term carbohydrate, fat and protein-based nutrition may adversely affect fertilization and embryo development due to abnormality in oocyte diameter.

In a study conducted by ZJ Lan et al. on mice, it was shown that overexpression of BMP15 and GDF9 genes caused hypofertility by decreasing the number of offspring and prolonging the estrous cycle [50]. Again, in another study by Y Peng et al. in mice, it was shown that BMP15 and GDF9 proteins play a heterodimeric role together and a very specific level of expression is required for normal oocyte development [47, 51]. These studies show that the decrease in the expression levels of these two proteins, as well as their higher than normal levels, negatively affect follicle and oocyte development. When the data in our study are evaluated, it is seen that there is an inverse correlation between the amount of BMP15 and GDF9 expression, and the number of advanced follicles and oocyte diameter. In protein group, while both BMP15 and GDF9 RNA levels are higher than the other groups, it is seen that all developing follicle types are the lowest in this group in terms of the number of follicles, excluding secondary follicles. This shows that the over-expression of BMP15 and GDF9 proteins may cause a decrease in the number of follicles inversely. In addition, in a study by JZ Lan et al., it is stated that overexpression of these proteins causes an increase in estrous cycle lengths [50]. When the estrous cycle data we obtained are examined, it is seen that the longest cycle duration is in the fat and protein groups, that is, in the groups with high BMP15 and GDF9 RNA levels. In other words, BMP15 and GDF9 proteins are expressed at a high level as a result of fat and protein diet, which negatively affects normal oocyte development by causing both a decrease in the number of follicles and a prolongation of the estrous cycle.

Considering the mechanism of action of Foxo3a, it is seen that it suppresses BMP proteins in primordial follicles and regulates follicle development by this mechanism [52]. According to the data we obtained, considering the Foxo3a mechanism of action, the decrease in Foxo3a expression in fat and protein groups results in an increase in the expression of BMP15 and GDF9, thus inducing reserve primordial follicles for further follicular development. Therefore, it is thought that the main molecular reason underlying the decrease tendency in follicle reserves in the fat and protein groups, may be the increased expression of BMP and GDF9 as a result of decreased Foxo3a protein. For that reason, the decrease in Foxo3a expression level as a result of unbalanced nutrition shows that it may cause both a decrease in the number of reserve follicles and inhibition of healthy follicle development in the ovary, resulting in infertility. The mechanism by which carbohydrate, fat and protein macronutrients suppress Foxo3a and whether they directly affect BMP15 and GDF9 independently of Foxo3a is a situation that needs to be clarified by new studies.

Besides many cells, GAS2 protein is also synthesized in oocyte and follicle cells, being more prominent in ovarian stromal cells, and is a necessary protein for normal follicle development [53]. Studies in Drosophila, pigs and mice have shown that GAS2 mutation results in sterility [28, 53]. The decrease in GAS2 expression we obtained in our study reveals another factor underlying the decreasing tendency in total follicle numbers in fat and protein groups. However, since the effect of GAS2 on folliculogenesis has been newly defined, there is not enough information about its mechanism of action yet [53].

The reduction in oocyte diameter at seconder and graafian oocytes in the carbohydrate group is thought to be related to the decreased expression of Zp1, Zp2 and Zp in this group. In addition, in the electron microscopic examinations (Supplementary Fig. 2), intense structural deformations were detected in the zona pellucida of the oocytes in this group. When we look at the relationship between the increase in the expression level of Zp1 and Zp3 genes in the protein group and the oocyte volume, it is seen that the oocyte diameter is abnormal from the primary follicle to the preovulatory follicle stage in the protein group. In other words, overexpression of Zp genes may cause anomalies in oocyte volume. In addition, intense structural deformations were detected in the zona pellucida structures, which were examined in electron microscopic sections of the protein group, similar to the carbohydrate group (Supplementary Fig. 2). It is thought that changes in the expression profiles of Zp proteins depending on the type of nutrition may also negatively affect fertility, for reasons such as the role of the zona pellucida in determining the oocyte volume, which has an important effect on fertilization by determining the borders of the oocyte, its role in sperm binding during fertilization and its functions in the prevention of polyspermia.

Increase in FSH and LH hormone levels in carbohydrate, fat and protein groups suggest that the induction of more primordial follicles for development in these three groups may be the reason for the decreasing trend in follicle reserve, respectively. In a study conducted by Chisato Nagata et al., it was shown that high fat intake increases the estrogen level [54]. In another study conducted by Akamine EH et al., it was stated that a high-fat diet increased the level of progesterone [55]. Considering the estrous cycle findings obtained in our study and the role of the estrogen hormone in the regulation of the estrous cycle, it is thought that the estrous irregularities and high abnormal estrous cycle numbers observed especially in the fat and protein groups may be the result of advanced synthesis of the estrogen hormone in those groups. As a matter of fact, studies have shown that the increase in progesterone and estrogen causes an increase in the length of the estrous cycle and disruption of the estrous cycle order [55].

Studies have aslo shown that adiponectin values ​​decrease, and resistin and leptin values ​​increase in obese animals [56]. In our study, adiponectin level was higher, resistin level was lower, and leptin level was similar to the control group, since the rats in the fat diet group did not reach the level of overweight or obesity yet. Leptin is known to be induce both sex hormones of pitiutary gland and ovary [57]. Similary, FSH, LH, E2 and progesteron were found to be increased in carbohydrate and protein groups in which leptin levels were also higher. One of the reason for induction of folliculogenesis especially in protein group may be due to high leptin induced over expression of genes such as BMP15, and GDF9, and hormones such as FSH, LH, E2 and progesteron playing role in folliculogenesis process. Studies have shown that changing the levels of adipose tissue hormones, the adiponectin, resistin and leptin, indirectly play a role in processes such as estrous cycle pattern, oocyte development and ovulation. However, there is insufficient information on how adipose tissue hormones affect nutrient consumption, which is important in oocyte development [56] and requires more extensive experimental studies.

Taken together, fat and protein-based nutrition cause estrous cycle irregularities, abnormal increase or decrease in the expression of folliculogenesis-specific genes and proteins, and changes in the levels of pituitary and ovarian hormones that have a direct effect on oocyte development. As a result of these changes, in the long term, the rapid decrease in primordial follicle and oocyte reserve, suppression of follicle development and decrease in oocyte quality may cause shortening of the reproductive period and decrease in fertilization potential. However, in order to fully understand the relationship between nutrition and fertility, more comprehensive and detailed experimental studies are needed to elucidate the mechanism-based relationships between nutrition types and changes in proteins and hormones involved in folliculogenesis.