Increased Risk of Primary Ovarian Insu ciency by High-Fructose Diet: A 90 Day Hormonal and Immunohistochemical Study in Wistar Rats


 There is ambiguous evidence that high-fructose diet can induce toxicity in different organ systems but its endocrine disrupting effects by abnormal changes in female reproductive organs is poorly evidenced. This study aimed to address the reproductive safety of high fructose diet through clinical, necropsy biochemical, hormonal, histopathological and immunohistochemical analysis. For this purpose, 5-6 weeks mature female Wistar rats were divided in three groups and each five animals/group exposed to standard chow+ water+ HFCS-55, standard chow+ water +sucrose 75 %w/v and standard chow+ water for 90 days. Remarkable increase in most lipid profile factors and total body weights of HFCS-55 fed rats and sucrose fed rats were detected in similar pattern compared to control .At the same time a battery of differential signs and symptoms in HFCS fed groups including destructive endometrial and ovarian changes , significant increase in FSH and LH levels, meaningful decreased serum testosterone and estradiol levels and strong AR expression in reproductive tissues of HFCS group of animals were recorded compared to other two study groups . These thought-provoking signs and signals of fructose induced reproductive toxicity in this model emphasis the contribution of HFCS-55 to deteriorated ovarian and endometrial health. More human population studies is necessary to find any possible association between fructose-rich diet and growing incidence of Primary Ovarian Deficiency (POI) in reproductive aged women .Further concern on this public health issue is required to revise existing regulatory standards and prevent this possible threat in human society.


Introduction
In the last four decades, severe changes in human nutritional patterns and higher consumption of high sugar rich diet have dramatically associated with increased incidence of hyperuricemia, dyslipidemia, hypertension, insulin resistance and overweight/obesity (Akarca-Dizakar SÖ, 2020) . General rising prevalence of obesity is highly associated with increased risk of endocrine disruption as evolving worldwide health crisis in adults and children (Zhou Y, 2021 ). A most recent animal study on rats shows a strong association between early exposures to high-sucrose diet and deteriorated ovarian health (de Melo GB, 2021).Higher incidence of total adiposity and truncal subcutaneous fat accumulation have become alerting women health problem with clusters of hormonal dysregulation which can lead to ovarian dysfunction (Ishikawa H, 2018). Prospective cohort studies indicate that routine consumptions of sugar sweetened beverages (SSBs) are closely associated with changes in hepatic metabolism and microbiome changes (Bhat SF, 2021),weight gain ,obesity , metabolic syndrome (Semnani-Azad Z, 2020), (Sadowska J, 2019 ) , (Collison KS, 2009 ), and increased risk of Polyc Cystic Ovarian Syndrom (PCOS) (Rizk MG, 2020) over time .Moreover neonatal exposure to SSBs may affect infant growth, predispose them to obesity phenotypes ,altered body composition (Michael I. Goran, 2017) and increased risk of ovarian toxic reactions in later stages of life .
Nowadays, fructose has become the main agent in sugar-sweetened beverages and sweets in many countries (Goncalves MD, 2019). Food industries prefer to use commercial high-fructose-corn-syrup 55% (HFCS-55) based on its sweetness, palatability , taste enhancement properties (DeChristopher LR, 2020) and taste maintaining properties of the beverages and food products (Chenxia Dai, 2020). Routine consumption of HFCS-sweetened beverage in breast feeding mothers increases the concentration of fructose in breast milk (Paige K. Berger, 2018) and uids from PCOS patients showed signi cantly higher fructose levels compared to healthy women regardless of whether the follicles were mature or immature (Shi B, 2020) .Based in this concept on female susceptibility to fructose induced changes we decided in the present work to nd any possible associations between HFCC-55 long-term consumption and any change in serum sex hormone levels ,ovarian and uterine histopathological changes and expression levels of sex steroid receptors by immunohistochemical method . We decided here to focus on HFCS-55 in females based on supporting evidence on sex speci c effect of fructose in women compared to men after high-fructose exposure (M. Rodgers, 2019)as well as the ubiquitous exposure of human populations to fructose through prepackaged foods, breakfast cereals, baked goods, dairy desserts, ssoft drinks and juice beverages (Zargaraan A, 2016).We compared the hormonal effects of HFCS-55 in original concentration(55%) with sucrose syrup 75% using OECD 408 guideline .

Study Subjects
High Fructose Corn Syrup 55 (HFCS-55) HFCS-55%, was kindly provided and certi ed by Zar Fructose Co., Ltd., Tehran, Iran in March 2020, under the code number of ZFQS04 with con rmed analytical sheet. According to analytical sheet, 100 ml of HFCS-55 provides 71.72 g carbohydrate (Fructose and Glucose), also the estimated energy of 100 ml of HFCS-55 is 286.9 Kcal.

Sucrose Syrup 75%
Puri ed and crystallized sucrose (saccharose) with over 99.7 ºZ polarization was provided and certi ed by Merck Millipore Co., Ltd., Germany under the CAS number of 57-50-1 with accessible analysis sheet in March 2020.Sucrose is made up of one molecule of glucose and one molecule of fructose joined together. It is a disaccharide, a molecule composed of two monosaccharaides: glucose and fructose.
Sucrose produces naturally in plants, from which, table sugar re nes. Sucrose is the most abundant disaccharide and the major product of photosynthesis. Sucrose syrup 75% was prepared by dissolving 75 g Sucrose with properties mentioned above in distilled water and adjusting up to 100 ml. We prepared each 100 ml of sucrose syrup 75% which provides about 280-290 Kcal energy.

Experimental animals and housing conditions
This study was carried out in a group of 15 mature regularly cycling female Wistar rats, aged 5-6 weeks, with initial body weights of 165-180 g which were purchased from Pasture Institute of Iran. Each 5 female rats were housed together in one standard cage in 12-h light/dark cycle (07:00-19:00), 23 ± 2°C temperature, relative humidity of 30-70%,8-15 times/h air change with access to tap water and standard diet ad libitum. The cage cleaning schedule, air ltration and recirculation, health checks and facility maintenance were carried out following to one-week adaptation and acclimation period.

Repeated dose oral toxicity study
The practical model of the experiment was conducted based on OECD 408 toxicology guideline.
After 10 days adaptation period, healthy female rats were randomly divided into 3 groups (5 rats/group) and labeled as Fo (HFCS-55), So (Sucrose 75%), and Co (control) groups. They were given free access to HFCS-water (HFCS group), Sucrose-water (Sucrose group) or deionized water (control group). Water and food consumption, general behavior and body weights were daily measured and recorded. Daily calorie intakes were calculated based on the calories levels in Table 1 and adjusted to the same levels based on the calorie levels obtained from ingested fructose or sucrose and standard rodents chow (3.34 kcal/g).
Clinical variables were considered as drinking and eating patterns, quality of response to environmental stimuli, surface of body reactions, body hair changes, drowsiness, any change in stool and urine colors, bringing up the tails, abnormal or ataxic gaits, any changes in eyes, salivation, changes in tear and total weight. At day 90 overnight fasted rats were sacri ced after blood collections by heart puncture under light carbon dioxide anesthesia and whole bloods were drawn for further biochemical analysis.

Biochemical Analysis
The collected blood samples were centrifuged at 1500 g for 15 min at 4 o C to obtain serums, and then the whole plasma were isolated and kept at -80 C until further analysis. The levels of glycemic factors were measured using an auto analyzer (HITACHI 917 / OLYMPUS AU640 & COBAS INTEGRA). Other than above factors. ,lipid pro le of animals was evaluated using serum concentration of total cholesterol, HDL, LDL and ratios using spectrophotometric enzyme assay kits (Amara Siri SS, 2020) in Bahar Toxicology Laboratory in Tehran.

Hormonal Assay
In order to determine the variations in serum concentrations of FSH, LH, Testosterone, Estradiol and progesterone , tail blood samples were collected according to standard protocol at day 15, 30,45,60, 75 and 90 of study and the serum was separated by cold centrifuge and collected in microtubes. Hormone Levels were compared with control by Chemiluminescence Immunoassays (CLIA) method using Cobas E411 from Roche Company with serial number of 15D3-16.

Recovery studies
A commercial rat serum pool (catalog no. M5905, Sigma Chemical Co., St. Louis, MO) was spiked with various hormone concentrations, and percent recovery and parallelism to the assay standard curve were determined. For each assay, the serum pool was spiked either with hormones across the assay range or vehicle to determine endogenous hormonal levels in the pool. Samples were run in duplicate, and each assay was repeated to con rm results. Hormonal recovery from each spiked sample was determined by subtracting hormone values in vehicle-spiked controls from hormone spiked samples.

Necropsy and Histopathological studies
During necropsy study, reproductive organs including uterus and ovaries were dissected out. Intact organs were rinsed and weighed with physiological serum to remove substances that may interfere with later stages and weighted. Organs were xed in 10% formalin solution at the next step. To remove the water, the desired tissue was dehydrated with degrees of alcohol (30, 50, 70, 80, 90 and absolute alcohol). To strengthen the xed tissues, they were placed in a para n blocks and nally 5 micron thin sections were prepared with a microtome. Multiple sections from each block were subsequently prepared at 5-micrometer diameters and stained with hematoxylin and eosin (H&E) for microscopically evaluations. The sections were examined under the light microscope (Olympus BX-51, Olympus, Tokyo, Japan) by expert animal pathologist and scored.

Immunohistochemcial Assay of sex hormone receptors
As previously described (Bidgoli SA ), (Bidgoli SA, 2011), dewaxed and rehydrated tissue sections were subjected to antigen retrieval using microwave oven and boiling citrate buffer (pH=6.0). Endogenous peroxidase activity and nonspeci c binding sites were blocked by incubating sections by 0.3% hydrogen peroxide in methanol for30 min and 3% BSA for 60 min, respectively. Sections were then incubated 30 min at Room Temperature with AR (Clone AR441, Dakocytomation) that recognize the nuclear expression of rat proteins in reproductive tissues. The results were visualized using Opti View DAB detection kit based on the manufacturer's instruction with necessary modi cations. Sections were also counterstained with Meyer's haematoxyline. In each series, a section in which incubation with the primary antibody was omitted used as negative control. The ideal staining conditions were established in our preliminary experiments. Staining was considered negative only after careful examination of the entire tissue section. All samples were assessed and scored by two independent pathologists. Histoscores of androgen receptor was assessed according to Intensity score and Allred score (P+I). In each case, a histoscore with a potential range of 0-300 was calculated as follows:

Statistical analysis
Study groups compared with one-way analysis of variance (ANOVA) and Post-hoc test. By student's t-test the difference between two sample means compared. Parametric values were expressed as mean ± standard deviation (SD) and the level of signi cance was set at p<0.05 in SPSS Statistics Software (version 21).

Clinical Effects
Clinical variables including quality of response to environmental stimuli, surface of body reactions, body hair changes, drowsiness, any change in stool and urine colors, bringing up the tails, abnormal or ataxic gaits, any changes in eyes, salivation, changes in tear were checked daily and remained unchanged until the end of study.
3.2. Total Body Weight: As described in table 1, the mean+ SD body weight was signi cantly increased in HFCS-55 fed group compared to control (267.62 ± 4.61 g vs. 230.52±0.84 g, p=0.0094). Similar change was recorded in Sucrose syrup fed group compared to Control (275.82 ± 6.6237 vs. 230.52±0.84 g, p= 0.0003). Table 1 presents and compares the mean serum levels of FSH, LH and sex hormones (estradiol, progesterone and testosterone) in female rats of the 3 study groups at day 15,30,45,60, 75 and 90.

Uterine Necropsy and Histopathology
As shown in table 1, the total weight of uterus was signi cantly increased in HFCS55-Fed group (1.4115±0.052 vs. 1.1975±0.050, p=0.0182) compared to control. The same difference was detected between HFCS fed animals and sucrose fed animals (1.4115±0.052 vs. 1.2102±0.047, p=0.0209) . HFSC intake was accompanied by destructive changes in the uterus of animals that means squamous metaplasia in uterine tissue of HFCS fed animals was detected (Fig 1 A) . Endometrial cells with apoptosis in HFCS fed animals (Fig1 B) and endometrial apoptosis with PMN cell in ltration and in ammatory changes in lamina propia in HFCS fed animals were the other detected destructive changes which observed.

Ovarian Necropsy and Histopathology
As shown in table 1, the total weight of ovaries were signi cantly increased in HFCS55-Fed animals (0.3528±0.070 vs. 0.2993±0.072, p<0.0001) compared to control. The same difference was detected between HFCS fed animals and sucrose fed animals (0.3528±0.070 vs. 0.3025±0.069, p<0.0001. HFSC intake was accompanied by destructive changes in the ovaries. The control and Sucrose-Fed groups demonstrated normal basic ovarian structure which usually contains corpus luteum and all developmental stages of follicles but moderate to severe congestion in ovarian tissue of HFSC fed animals were observed (Fig 1B1). Table 2 compares the expression levels of Androgen Receptor (AR) based on Propotion score (P), intensity score (I) and Allred score (P+I) in Epithelial cells of ovary follicles and Ovarian super cial epithelium.AR was overexpressed in ovaries of HFSC fed animals according to Propotion Score, AR Intensity Score and allred score but similar to control group, the expression pattern of AR remained unchanged in sucrose fed animals.

Uterine Expression of Androgen Receptor (AR)
Mild Nuclear expression of AR in endometrial epithelium of HFCS fed animals ( Fig 2D) and moderate nuclear expression of AR in endometrial epithelium of Sucrose fed animals (Fig 2E) was detected .AR expression in endometrial epithelium of control group was strong (Fig 2F). Table 2 compares the expression levels of Estrogen Receptor (ER) based on Propotion score (P), intensity score (I) and Allred score (P+I) in Epithelial cells of ovary follicles and Ovarian super cial epithelium.ER expression pattern was practically similar in ovaries of study groups.

Lipid pro le
As shown in table 3, all lipid pro le factors in HFCS-55 fed rats signi cantly increased compared to control and Sucrose groups except HDL-Cholesterol. Out of different lipid pro le related factors, LDL-Cholesterol and LDL/HDL ratio raised signi cantly just in HFCS-55 fed animals (p=0.008) not in sucrose group. Other factors changed in the following manner: Triglyceride (TG mg/dL): Serum level of TG in HFCS-55 was signi cantly higher than control (p<0.0001) and Sucrose (p=0.0034).
Serum level of total Cholesterol raised signi cantly in both treatment groups compared to control but the difference was higher in HFCS-55(p=0.0095) than Sucrose (p=0.045).
Non-HDL Cholesterol also increased signi cantly in both treatment groups compared to control but the difference was again higher in HFCS-55(p=0.0021) than Sucrose (p=0.0062).
Cholesterol/HDL ratio was increased in both groups but the level in HFCS-55 fed animals was signi cantly higher (p=0.0165).

Glycemic factors
As shown in table 3, out of different glycemic factors, FBS increased in both study groups but the mean (SD) was signi cantly higher in HFCS-55 compared to control (125±17.21918 vs. 79±15.033, p=0.002) and the difference with Sucrose was not meaningful (p=0.6302).

Discussion
Global prevalence of primary ovarian insu ciency (POI) is continuously growing with partially or complete cessation of ovarian function (Golezar S, 2019) but more than one fourth of Iranian women experience early menopause before 45, especially the non-normal weight ones and this high prevalence should be considered as a critical public health concerns that needs to be addressed by health policy makers (Marzieh Rostami Dovom, 2021). Our recent study on the contribution of dietary factors in ovarian toxicity (Bidgoli SA, 2021) has encouraged us to focus on the role of other routine nutritional parameters in ovarian dysfunction and selected fructose-rich diet for this purpose . To achieve this goal, we tried in this 90 days study to compare the reproductive effects of HFCS-55 enriched normal chow diet with two control groups (normal chow diet and sucrose enriched normal chow diet) through clinical, biochemical , hormonal ,histopathological and immunohistochemucal analysis. In the context of many expectable changes in total body weight, glycemic factors and lipid pro le in both fructose and sucrose fed animals (table 2) , differential toxic reactions in HFCS-55 fed animals were accompanied by destructive changes in ovaries and uterine according to necrospsy,histopathologica and immunohistochemical changes compared to sucrose fed animals . Highly signi cant increase in FSH and LH levels, meaningful lower serum testosterone and estradiol levels and extensive AR overexpression in reproductive tissues of HFCS group of animals are thought-provoking signs and signals of fructose induced reproductive toxicity in this model which is discussing below.
Development of hyper-gonadotropic hypoestrogenism signs and symptoms in women before 40 years of age is referred as premature ovarian failure (POF), or "premature ovarian insu ciency" (POI) (Marzieh Rostami Dovom, 2019). The idiopathic trio of POI could be associated with increased risk of idiopathic infertility, cardiovascular disease (CVD), decreased bone mineral density (BMD), vulvovaginal atrophy, psychological distress, neurological effects and overall reduced quality of life and life expectancy (Tsiligiannis S, 2019), (Rossetti R, 2017).Through animal studies ,normally mature Rats with fully developed hypothalamic-pituitary-gonadal (HPG) axis and serum androgen-estrogen levels are susceptible to xenobiotic induced ovarian dysfunction and our ndings clearly con rmed this sensitivity to dietary high-fructose intake in female rats .Female rat susceptibility to high-fructose diet was con rmed by higher LH and FSH levels (p<0.001), lower serum testosterone levels (p=0.0032), lower serum estradiol levels (p<0.001),increased total body weight(p=0.0094) , higher levels of serum lipids (p=0.001) and increased FBS (p=0.002) compared to control groups. This pattern was partially described elsewhere in male rats with high fructose diet before (Tkachenko OY, 2020). Based on these ndings further epidemiological studies seems necessary to nd any possible association between high-fructose diet and POI in Iranian /middle east women espceially in overweight cases .
The endometrium is a complex multicellular tissue which is extensively sensitive to the synthesis and release of sex steroids in the ovary (Gibson DA, 2020), a process which was obviously disturbed in our HFCS-55 fed animals .The structural impact of estradiol and testosterone de ciency on endometrial tissue was accompanied by uterine metaplasia , apoptotic changes and initial evidence of in ammation by Neutrophil (PMN) in ltration which plays a central role in in ammation and could be considered as a major cause of tissue damage. These battery of changes are initial evidence for possible later clinical diseases especially infertility, endometriosis (Vercellini P, 2014) and reproductive malignancies (PP., 2018) by summarizing evidence from studies on these issues. Based on these preliminary ndings with signi cant uterus weigh in HFCS-fed animals further epidemiological studies seems necessary to nd any possible association between high-fructose diet and endometrial malignancy,infertlity and endometriosis especially in overweight ,hyperglycemic or hyperlipidemic women .
The ovary, is one of the main sources of androgen production in women. A detailed evidence of Androgen Receptor overexpression in ovary and endometrial tissues of HFCS-55 fed animals in parallel to testosterone de cient are good evidence which was associated with ovarian congestion in this group of animals but further studies on sex hormone binding globulin (SHBG), free androgen index (FAI)(Santoro N, 2011) in animal and epidemiological studies seems necessary. We predict also overexpression of ER in longer study design according to signi cant estradiol de ciency in present study and PR downregulation based on overproduction in this study setting.

Conclusion
Using veri ed OECD guideline (TG 408) and evaluation of sub chronic oral toxicity of HFCS-55 commercial samples in original dilution in an ad libitum oral 90 days intake model, suggests the hypothesis that highfructose diet can induce primary ovarian insu ciency , uterine metaplasia and in ammation by meaningful hormonal, histopathological and immunohistochemical evidence. We strongly believe that reproducibility of present results should be assessed in similar setting using HFCS-55 derived food products. Based on recent published literature on toxic effects of high-fructose diet and present study results on differential role of HFSC-55 on female reproductive and hormonal functions , continued use of HFCS-55 in the food industries with existing governmental standards and requirements may need some revisions . Human population studies in exposed populations is also necessary to understand possible associations between growing incidence of POI and dietary fructose as a very important public women health issue in parallel to more experimental studies to de ne new necessary policies in the future .

Declarations
Acknowledgements The authors are thankful from Zar Fructose Co., Ltd., Tehran, Iran for providing our study samples. The authors also acknowledge Dr. Hasti Azar Abad as the veterinary pathologist of the present study for her excellent supports of histopathological studies.We acknowledge cancer Institute of Iran for supporting immunohistochemical analysis .

Author contribution
Roya Mirzaei performed all animal studies and lab as part of her PhD thesis. She read and approved the manuscript before submission. Sepideh Arbabi Bidgoli is the main supervisor of this study who planned, designed the work, conducted, performed the statistical analysis, and provided the manuscript. Roya Khosrokhavar was the second supervisor of this PhD study who suggested the main topic and contributed in planning and study design execution of HFCS-55. Shahram Shoeibi and Hamidreza Ahmadi Ashtiani were co-advisors of this PhD program who were contributed to all parts of this study.
Availability of data and materials: All data and materials are available upon journal request.