The impact of non-caloric artificial sweetener aspartame on female reproductive system in mice model

Background Artificial sweeteners, used as sugar substitutes have found their ways into almost all the food items due to the notion that they are non-caloric. Aspartame is used in numerous food products throughout the world. The primary users of aspartame include diabetics and calorie conscious people who intend to limit their calorie intake. Methods Female Swiss albino mice were divided into three groups (12 mice each) for the duration of 30 and 60 days consecutively. The treatment groups received 40 mg/kg b. w. aspartame orally. Hormone assays using ELISA and tissue histopathology have been performed along with the fertility assay to access the treatment outcomeon the fertility of treated mice in comparison to controls. Results Present study reports that female mice treated with aspartame for 30 and 60 days showed significant reduction in body weight, relative organ weight of (liver and kidney) and gonadosomatic index. These changes were more significantly recorded in 60 days treatment group. Aspartame treated animals for 30 and 60 days showed duration-dependent decrease gonandotropins (follicle stimulating hormone and luteinizing hormone), and steroids (estradiol and progesterone). Moreover, severe histopathological changes, reduction in number of growing follicles, degenerative changes in follicular structure, corona radiata and zonagranulosa were also observed. Besides, histomorphological changes were also observed in the uterine structure including atrophic uterine endometrial glands, contracted endometrial lining, disruption of the endometrial structure and the shapes of blood vessels were also altered. Conclusion Non-nutritive artificial sweeteners including aspartame negatively impact the function of ovaries and feedback mechanism of reproductive hormones by affecting the hypothalamic–pituitary–gonadal axis. In light of present findings the aspartame negatively impacted the reproductive system of female mice. More studies are required to identify the molecular mechanism and the pathways involved.


Background
Humans and other animals naturally like sweet tastes, which boosts eating satisfaction [1,2].Sugar is bad for teeth, waistline, and is associated with several degenerative disorders.Sugar intake may affect behaviour, emotions, health, and disease [3][4][5].The number of people suffering from diabetes [6], obesity [7], hypertension [8], and heart disease [9] is increasing every year.Increased sugar in foods, desserts, and drinks has prompted health concerns.Added sugar intake has increased worldwide obesity [10].The World Health Organization recommends that added sugars should make up no more than 10% of daily caloric intake for optimal health [11].Likewise, American Heart Association (AHA) recommendation is 6 table spoon full (24 g, providing 100 calories) of sugar per day for women and 9 table spoon full (36 g, providing 150 calories) of sugar per day for men [12].
The popularity of sugar free foods is attributed to their low calorie content.A number of low calorie artificial sweeteners acesulfame K, aspartame, neotame, saccharin, stevia, and sucralose [13,14] having calorie value lower than sugar are produced throughout the world in a very large quantity.The admissible daily intake (ADI) for aspartame is 40 mg per kilogram body weight (for humans) as stated by FDA.However, this value has given birth to a number of controversies, wherein, many researchers have reported that not only the ADI but lesser quantities are not totally safe for consumption [15,16].A number of health ailments like metabolic syndrome, increased weight gain and other negative health effects have been associated with sugar intake [17][18][19][20].This has finally resulted into the promotion of ASs as a healthy alternative [13,21].Sugar substitute is an artificial non-nutritive sweetener which mimics the effect of sugar on taste [22].Artificial sweeteners (ASs) are also known as non-nutritive or intense sweeteners because the sweetening potential of ASs is very high compared to common sugar.Aspartame containing foods provide a kind of dietary option which is considered helpful in containing obesity or diabetes mellitus [23].
However, ASs has been associated with numerous adverse effects [24,25].Many studies have investigated the impact of ASs exposure during pregnancy and early childhood but due to the obscure conclusions of the studies about the impact of the ASs during critical developmental periods adds to the controversy [26][27][28].According to the American Dietetic Association ASs consumption is safe in children and pregnant women within acceptable intake limits [13], however, the US Institute of Medicine states a paucity of evidence of ASs safety and suggests avoiding ASs use in childhood [29].Food and beverage intake of non-nutritive sweeteners (NNSs) has increased worldwide over the last three decades.Consumers' preference of NNSs rather than sugar or other healthy sweeteners might be due to their ability to minimize weight gain [30].ASs are so abundant and widespread in the food industry that a number of people even do not know that they are consuming them [31].On the basis of some studies supporting the use of ASs, if their use for human consumption is deemed safe [32,33], significant evidences suggest that ASs may not be necessarily healthy, do not mitigate weight gain and may not be good to improve circulating glucose levels [34].
Gut-brain axis plays a vital role in sensing of foods ingested by humans.Feedback circuits are initiated by this axis to modify gene expression and regulate glycemia, satiety, and energy partitioning [35,36].Aspartame (ASP) is one of the most commonly used ASs and is used as sugar substitute in a number of food products including, soft drinks, jams, chewing gum, canned fruit, candies, cosmetic products, vitamins, and medications [37,38].There are hundreds of millions of aspartame consumers throughout the world.Children and women of child bearing age are the major users of ASP [39,40].The consequences of ASP intake in pregnant women have been minimally addressed.A few studies investigating the impact of ASP on the gestation in humans are available.Besides, a few studies, in which the effect of ASP on the weight and physiology of offspring have been studied, are also available [41,42].

Animal model and aspartame administration
The animals used in this study were maintained in the animal centre of the Department of Biosciences in accordance with the Institutional Animal Ethical Committee (IAEC) and Committee for Control and Supervision of Experiments on Animals(CCSEA), New Delhi, India, (No. 1885/GO/S/16/CPCSEA/IAEC//B.U./08 Dt. 18/06/16).The female mice were housed in standard polypropylene mice cages (290 × 220 × 140 mm) containing rice husk as bedding material.The animal room was well ventilated and maintained under standard experimental conditions (Temperature 22 ± 2ºC and 12 h light/ dark cycle) throughout the experimental period.All the animals were provided with standard pellet diet and water ad libitum.The animals were acclimatized to the standard laboratory conditions for one week prior to experimental use.Healthy female albino mice Parkes (P) strain (5 to 7 weeks old) of 20 ± 2 g body weight was used in this study.Twelve mice were kept in the each group among, six were used for fertility assay and six were sacrificed for serum and tissue analysis for each duration.The animals were purchased from the College of Veterinary Sciences, Mahow, Indore, Madhya Pradesh, India.The animals were randomly assigned to each experimental group.Aspartame (C 14 H 18 N 2 O 5 , 99% pure CDH-Laboratory Chemicals India) was purchased in powder form and mixed in water to make it suitable for the oral ingestion.The control groups received distilled water by oral gavage and aspartame groups received 40 mg/kg b. w./ day aspartame (2 mg/ml/2000 ppm) dissolved in distilled water for 30 and 60 days, until the completion of the study.The dose and duration of aspartame (ASP) used in the present study were based upon the previous studies [42][43][44].At the end of the experiment, six female mice from each group were used for analysis or relative organ weights (liver and kidney), GSI, hormonal analysis and histopathological examinations of ovaries and uteri and another six animals were used for fertility studies.

Fertility assessment
After the aspartame treatment for 30 and 60 days the females were housed with virgin untreated males in the ratio of 2:1.The males were removed after 24 h and the successful mating was confirmed by the presence of vaginal plug.The females were observed for successful pregnancy, fertility rate, gestational length, litter size, litters weight [45].

Serum sampling and processing
At the end of 30 and 60 days aspartame administration, the female mice were sacrificed and blood samples were collected by cardiac puncture and centrifuged at 3000 rpm for 10 min to obtain serum.The serum samples were further used for the analyses of luteinizing hormone(LH), estradiol (E2), progesterone(P4), and follicle-stimulating hormone(FSH) levels.

Body weight, relative organs weight, and gonadosomatic indices (GSI)
The body weights of the experimental animals, control as well as treatment were recorded at the initial day i.e. zero days and at the end of the different durations of the experiment i.e. 30 and 60 days.The values were expressed in grams [46].
The body weight gain was calculated as: Body weight gain = Final body weight-Initial body weight.
Whereas, relative organs weight (liver and kidney) was calculated as: Relative organs weight = weight of organs (g) ÷ Final weight x 100.

Estimation of Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Progesterone (P4), and Estrogen (E2) in the serum of female mice
The levels of E2, P4, FSH and LH were detected using mouse specific enzyme-linked immunosorbent assay (ELISA Kits) (Calbiotech Inc CA United States), according to the manufacturer's recommended instructions.

Histopathology
Animals of the treatment as well as control groups were sacrificed at the end of the experiment i.e. 30 and 60 days duration.Liver, kidneys, ovaries and uterus were dissected out immediately after the animals were sacrificed, washed in cold 0.9% NaCl, cleared of any attached tissues and fats, and were blotted dry.Small sections of all the organs were placed into separate vials containing Bouin's fixative.All the vials were labelled with name of the organ, date of fixation and group details. 5 µ thick paraffin embedded tissue sections were cut, and stained with Ehrlich's Hematoxylin and Eosin [48].The stained sections were observed under compound microscope at 100X and 400X magnifications for any histopathological changes.The microphotograps of the observed tissue sections were taken by using microphotography unit.

Statistical analysis
The observations and data were tested for statistical significance using SPSS software.The values were expressed as mean standard deviation (Mean ± SD).One way ANOVA is performed to analyse the statistical differences among groups.p < 0.05 is considered significant.

Body weight
The animals were weighed initially and after different intervals i.e. 30 and 60 days of the aspartame experiment.
The animals treated orally with ASP showed a significant (p ≤ 0.01) decrease in their body weight when compared to the respective controls.The decrease in body weight was significant (p ≤ 0.001) in duration dependent manner (Table 1).

Relative organs weight and gonadosomatic indices
After treatment of ASP for 30 and 60 days, the relative organ weights of liver and kidney were decreased significantly (p ≤ 0.01) in ASP treated group when compared to control groups.Decrease in relative organ weights of liver and kidney was highly significant (p ≤ 0.001) in 60 days ASP treated group.There were no significant changes in relative organ weights in control groups.The ovarian weights reduced significantly (p ≤ 0.01) after the treatment of ASP for 30 and 60 and days, as compared to control groups.The reduction in ovarian and uterus weight was significant (p ≤ 0.001) in 60 days of ASP treated group (Table 1).

Hormone analysis
Animals treated with aspartame for 30 and 60 days showed a significant (p < 0.01) decrease in FSH and LH hormonal levels as compared to control group.The decrease was significant (p < 0.001) in 60 days treated group.In addition, estradiol (E2) and progesterone (P4) levels also showed a significant (p < 0.01) decrease in 30 and 60 days of ASP treated groups when compared to control groups (Fig. 1).

Follicle count and fertility parameter
Females treated with ASP showed significant (p ≤ 0.05) duration-dependent decrease in follicle count compared to control including primordial and growing follicles.Besides, animals treated with ASP showed duration dependent decrease in litter size, body weight of pups postnatal viability, weaning index and fertility index compared to control group.However, no significant difference was observed in gestation period in 30 and 60 days ASP treated groups compared to control (Tables 2 and 3).

Histopathological and histomorphological analysis
The ovary female mice of the control group showed normal histoarchitecture including normal follicles, different sizes and stages of developing oocyte, zona granulose and thecal layers of follicle and corpus luteum (Figs.2a and 3a).However, the female mice exposed with ASP for 30 (Figs. 2b and 3b) and 60 days (Figs.2c  and 3c) showed histopathological changes in the ovarian structure characterized by decreased number of growing follicles, degenerative changes were observed in follicular structure i.e. degenerating oocytes, theca layers, corona radiata and zonagranulosa.The connective tissue of medullary region also showed some degenerative changes and vacuolization. .Female mice of control group presented a normal uterus histoarchitecture with normal endometrium, endometrial glands, myometrium, intact perimetrium and simple columnar epithelium (Figs.2d and 3d).Animals treated with aspartame for 30 days (Figs.2e and 3e) and 60 days (Figs.2f and 3f ) presented histomorphological changes in the uterine structure including atrophic uterine endometrial glands, squeezed endometrial lining, disruption of the endometrium and the shapes of blood vessels were also altered.

Discussion
The extensive use of artificial sweeteners particularly aspartame and its associated negative effects have been investigated by many researchers.However, reproductive toxicity of aspartame has been minimally addressed.
The results of the present study showed that ASP administration induced duration dependent decrease in body weight.The reduction in body weight was significant (p < 0.001) in 60 days treatment group as compared to the control.The reduction in body weight may be due to reduced food and water intake which may have been caused by ASP consumption.These observations are supported by some researchers [49,50] who reported that ASP intake induces satiety, decreases food intake and bodyweight.ASP administration increases circulating blood levels of phenylalanine which is reported to suppress food intake in humans and animals and increases cholecystokinin secretion which delays the gastric emptying [50][51][52][53].ASP was also reported to reduce body weight and fat mass in overweight subjects [50,[54][55][56].Some researchers believe that decreased body weight in ASP treated animals is a result of diminution of Neuropeptide Y (NPY) in its principal hypothalamic site of synthesis [57].Neuropeptide Y (NPY) inhibits lipolysis and stimulates de novo lipogenesis and thus promotes weight gain and fat deposition [58][59][60][61].
The relative organ weight and GSI of animals treated with ASP significantly (p < 0.01) decreased after 30 and 60 days of ASP administration.The observed decrease in relative organ weight and GSI were significant (p < 0.01) in 60 days ASP treated group as compared to the control group.The measurement of relative organ weight and GSI is an important indicator to study the organ toxicity due to the exposure of any toxic chemical.The durationdependent decrease in relative organ weight and GSI suggest abnormalities and atrophy in these organs.The results observed suggest that ASP may have toxic effects on liver, kidney and ovary which may have been caused by the methanol intoxication that increase the lipid peroxidation (LPO) [62,63].It was reported that aspartame can act as chemical stressor by increasing corticosteroid level which in turn has been shown to decrease the size and weight of organs due to oxidative damage [64,65].
Formaldehyde, which is the first metabolic product of methanol, increases the population of shrunken and dead cells [66][67][68] which might be responsible for decreased organ weight.
Aspartame treated animals for 30 and 60 days showed duration-dependent decrease in androgens (FSH and LH), and steroid (E2 and P4) hormone levels.The decline was significant (p < 0.001) in 60 days of ASP treated group as compared to the control group.We also observed marked histomorphological changes in ovary and uterus of the ASP treated female mice in duration dependent manner.Female mice treated with ASP showed many histomorphological changes in the ovarian structure including decreased number of growing follicles.Degenerative changes were observed in follicular structure like degenerating oocytes, theca folliculi, corona radiata and zona granulosa.The connective tissue stroma and medulla region also shows some degenerative changes and vacuolation.Aspartame has markedly damaged the reproductive function of the female mice as is evident from the alterations in hormone levels and histomorphological changes in ovary and uterus.The toxic effect of ASP on the reproductive performance of female mice is also manifested by significant (p < 0.05) decrease in pregnancy rate, litter size, litter weight and viability index.This effect may have been caused by uterine and ovarian abnormalities which may have been a direct result of ASP exposure.The ovary is an important target organ of many toxic chemicals and neuroendocrine disruptors [69][70][71][72].We observed that both ovarian and uterine weights were significantly (p < 0.01) decreased in ASP treated animals.These results suggest that ASP may have toxic effects on both ovarian and uterine structures.There are not many studies available on the effect of ASP on gonadotropins and ovarian steroid hormones.A few studies have investigated the effect of ASP on male reproductive system including testosterone hormone and histology of testes, sperm quality, viability and motility [73][74][75].A significant decrease in total antioxidant capacity (TAC) and testosterone along with significant increase in malondialdehyde (MDA) was reported following ASP treatment.Aspartame administration decreased number, motility, viability and maturation of sperms along with increased abnormality and DNA damage to sperms [73,76,77].Aspartame administration decreased number, motility, viability and maturation of sperms along with increased abnormality and DNA damage to sperms.Aspartame is reported to have deleterious effects on hypothalamus which produces gonadotropin releasing hormone (GnRH) which goes down the pituitary stalk stimulating the pituitary gland to produce gonadotropins stimulating the testicles and ovaries to produce testosterone [78,79].Aspartame is metabolized in the gut releasing the aspartic acid, phenylalanine, methanol, and diketopiperazine.Methanol is converted into formaladehyde and formic acid above 85 o F and is said to have toxic effects on central nervous system (CNS), gatro-intestinal (GI) tract, liver, kidney and testes [80].The toxic effects of aspartic acid and methanol on testes are due to the potential of these two components to cross blood-testes barrier and reduce spermatogenesis, reduced tubule size, spermatogenic arrest, and inhibition of steroid biosynthesis in Leydig cells which is an outcome of oxidative stress [81][82][83][84].
Chronic administration of ASP (2 mg/g b. w.) induced selective degeneration of all subcelular neurons ultrastructures both in CA1 pyramidal neurons of hippocampus and in ventral-medial area of hypothalamus, which control the activity of pituitary and intense vacuolization like damages [79,85], loss of the intracytoplasmatic secretory granules in all cellular ultrastructures of the adenohypophysis and therefore alter the homeostasis [86].Electron-microscopy observations revealed that ASP treatment induced marked alterations in growth hormone (GH) and LH/FSH cells in rats.These alterations were more prominent in either GH or LH/FSH secretory cells, indicating a decline in growth and gonadotropic hormones secretion.Besides, young prepubertal rats appear to be most susceptible to the deleterious effects of ASP [87,88].Aspartame induced lesions of the mediobasal hypothalamus are associated with a low release of gonadoliberins and low level of gonadotropic hormones including inhibition of the synthesis and secretion of testosterone.All these changes have an overall effect on the diminution of the reproductive capacity [79].These observations are in agreement with the finding which shows that administration of excitotoxins (glutamate, aspartic acid, cysteine, and their homologues) to lab rodents through different routes caused neuronal degeneration, associated with axonaldendritic lesions [89][90][91].
Some studies showed that increased concentrations of ASP metabolites like phenylalanine, aspartic acid, and methanol are responsible for alterations in hormone levels [67,92,93].Increased concentrations of aspartame metabolites were reported in blood following ASP consumption [94].Excessive phenylalanine interferes with the tyrosine and tryptophan resulting in decreased concentrations of the brain catecholamine, serotonin and dopamine disturbing the balance of neurotransmitters.This in turn leads to neurological, behavioural and hormonal changes [67,68,94].Granulosa cells and follicular membrane cells in the follicle are the target cells of steroid hormones and gonadotropin [95,96].
We also found that the ovarian and uterine weights were significantly decreased in ASP treated groups.In addition to this, histomorphological changes in both ovary and uterus were also observed in ASP treated groups after different intervals.The changes induced in the ovarian structure after ASP administration characterized by decreased number of growing follicles, degenerative changes were observed in follicular structure i.e. degenerating oocytes, theca layers, corona radiata and zona granulosa.The connective tissue of medulla region also showed some degenerative changes and vacuolization.Besides, ASP also induced histomorphological changes in the uterine structure including atrophic uterine endometrial glands, strained endometrial lining, disruption of the endometrium and the shapes of blood vessels were also altered.Steroid hormones like estradiol (E2) and progesterone (P4) play a very important role in the growth and differentiation of reproductive tissues and maintenance of fertility [97,98].Estrogen enhances the sensitivity of granulose cells to FSH and LH, thereby increasing the biosynthesis of progesterone by granulose cells [97,99].Estrogen modulates steroidogenesis, promotes granulose cell proliferation and maintains follicular development [100][101][102].The release of LH and FSH from the anterior pituitary gland regulates the secretion of reproductive hormones from the ovary [103,104].The main function of FSH includes stimulation of ovarian growth and promotion of follicular development.Luteinizing hormone plays an important role in follicular maturation, ovulation, corpus luteum development and is involved in the synthesis of steroid hormones [105][106][107][108][109][110].A decreased tendency of females to get pregnant could be due to decreased levels of reproductive hormones.We observed that the endometrium was damaged, and the endometrial glands were atrophied which may be the cause of low fertility rate of ASP treated groups.It is reported that endometrial completeness is critical for the successful implantation of an embryo [111,112].Apart from this, a significant decrease in litter size was observed in our study along with decreased body weight of pups in all ASP treated groups.The significant change in gestation period, viability index and weaning index in 90 days ASP treated group.The reduction in body weight of pups is attributed to insufficient availability of substrates including glucose to the foetuses due to possible diminution of substrates in the ASP fed maternal blood [113][114][115][116].It was observed that methanol formed during the ASP metabolism might be responsible for preterm delivery as methanol has been shown to decrease gestational length in primates [65,[117][118][119].

Conclusion
In light of the present findings ASP has a correlation with the possibilities of reproductive toxicity.The study conclude that aspartame and its metabolites have the potential to affect female reproductive systems, gestation period and fetal development and pregnancy outcomes.We propose that ASP affects hypothalamic-pituitarygonadal axis (HPG axis) altering the release of LH and FSH from the anterior pituitary gland and damages the histomorphology of ovary and uterus like follicular maturation, ovulation and corpus luteum development.It is also concluded that uterine endometrium abruption and the atrophy of the uterine glands were the result of ASP intake.ASP decreased the tendency of animals to get pregnant by deminishing the levels of gonandotropins.
Small littersize,decreased fetal weight.and extended gestational period supports the conclusion.That aspartame intake should be taken seriously.Aspartame related research investigations are further advised to identify mechanism and pathways affected by ASP consumption and its metabolic breakdown products to understand the molecular mechanism of reproductive alterations and related disorders progression.

Fig. 2
Fig. 2 (a) Section of ovary of control mice showing normal growing follicles (F) of different sizes and stages.(b) Section of ovary of mice treated with ASP for 30 days showing decreased number of growing follicles.Degenerative changes were observed in follicular structure like degenerating oocytes (DO), theca follicli (arrow), corona radiata (arrow head) and Zona granulosa (ZG).(c) Aspartame treated ovary for 60 days shows degeneration of follicular antrum (FA), corona radiate (red arrow), oocyte (O), zona granulose (ZG) and theca follicle (arrow head).The number of follicles is very less.(d) of control uterus showing perimetrium (P), myommetrium (M), and endometrium (E).(e) Section of uterus of mice treated with ASP for 30 days shows compressed endometrial cells (arrow heads) and the glands (arrows) are also atrophic, attributed to endometrium destruction due to ASP.(f) Uterus treated with ASP for 60 days shows degeneration in uterine endometrial lining (arrow head).Endometrial glands were also affected as their number was decreased and their epithelial lining showed necrosis (arrows) (H & E × 100)

Fig. 3
Fig. 3 (g) Magnified view of control ovary section showing developing follicles with normal histological features characterized by well defined granulose cells (GC) surrounding the oocyte.(h) Section of ovary treated with ASP for 30 days shows degenerative changes in follicles (DF), degeneration of follicular antrium (FA), primary Oocyte (O), theca follicli (thick arrow), corona radiata (arrow head) and zona granulosa (ZG) and decreased number of primary follicles.(i) Section of ovary treated with ASP for 60 days shows degenerative changes in thecal layer of follicle membrane (arrows), zona granulose (ZG), and zona pellucida (arrow head).The appearance of pyknotic bodies (atretic bodies) in granulosa cells (star) and granulation of cytoplasm are the indications of early follicular atresia.(j) Uterus of control mice showing normal endometrium with stratum basale and stratum functionalis bearing fibrous connective tissue and normal tubular glands (arrows).(k) Uterus treated with ASP for 30 days shows atrophic uterine endometrial glands (arrows), squeezed endometrial lining (arrow head) and the shapes of blood vessels were also altered (arrows).(l).Uterus treated with ASP for 60 days shows larger endometrial cells (arrow head) and atrophic endometrial glands (arrows).Myometrium (M) also appears atrophic due to atrophy of the smooth muscle cells (H & E × 400)

Table 1
Body weight (g), relative organ weight (g/100 g b. w.), and GSI (mg/100 g b. w.) of control and aspartame (ASP) treated female mice after 30 and 60 days *Significant difference (p ≤ 0.05) compared to control by one way ANOVA **More significant difference (p ≤ 0.01) compared to control by one way ANOVA ***Highly significant difference (p ≤ 0.001) compared to control by one way ANOVA NS Non significant compared to control by one way ANOVA

Table 2
Follicular count (Ovarian reserves) of Control and Aspartame treated mice after 30 and 60 days **More significant difference (p ≤ 0.01) compared to control by one way ANOVA ***Highly significant difference (p ≤ 0.001) compared to control by one way ANOVA NS Non significant compared to control by one way ANOVA

Table 3
Reproductive indices of Control and Aspartame (ASP) treated female mice after 30 and 60 days ± SD of six animals *Significant difference (p ≤ 0.05) compared to control by one way ANOVA **More significant difference (p ≤ 0.01) compared to control by one way ANOVA ***Highly significant difference (p ≤ 0.001) compared to control by one way ANOVA NS Non significant compared to control by one way ANOVA