This study was carried out in full accordance with the ethical guidelines set by the Animal Care Committee, ensuring the highest standards of animal experimentation ethics were upheld. The experiment involved 32 adult female Sprague Dawley (SD) rats, which were accommodated in the specialized Animal House Unit at the Iraqi Center for Genetics and Cancer Research. The rats were provided with an environment that was optimized for their well-being, with a stable temperature of 23 ± 1°C and a balanced light-dark cycle of 12 hours each. The SD rats had unrestricted access to food and water throughout the study.
Before the experimental procedures began, a two-week acclimation period was observed to allow the rats to adjust to their new environment and minimize stress. The SD rats were carefully divided into four distinct groups, each consisting of five rats (n=8). The groups were allocated as follows [30–32], the first group served as the control and did not receive any treatment. The second group was treated with AuNPs, receiving a daily dose of 200 mg/kg administered via gavage. The third group was given a daily dosage of 10 ml/kg of green tea extract, also administered via gavage. The final group received a combined treatment of both AuNPs and green tea extract, with the dosages and administration method consistent with the individual treatment groups. 
The SD rats underwent a treatment regimen spanning 28 days, with treatments administered at 24-hour intervals. Following the completion of this phase, the rats were first anesthetized using diethyl ether to ensure a painless procedure. A careful dissection was then carried out, during which the left ovaries were extracted with precision. The extracted ovaries were weighed to document their mass, providing crucial data for the study. To preserve their structural integrity for subsequent analysis, the ovaries were immediately placed in Bouin solution.
After a fixation period of 18 hours, the tissue samples were subjected to a series of meticulous processing steps. This included the transition of tissue through various stages and the creation of paraffin blocks using cylindrical molds. These procedures were executed in strict accordance with the principles of Independent Uniform Random (IUR) sectioning [33]. This method, renowned for its capacity to produce sections that are random across all three planes, is particularly invaluable in the realm of stereological studies.
This involved a two-step randomization process using ф and θ coordinates. Each ovary was placed on the ф coordinate system, represented as a clock face. A random number between 0 and 9 was selected from a numerical table to determine the cutting angle. Based on this number, the ovary was bisected, resulting in two separate pieces. The first piece of each ovary was then aligned on the θ clock, ensuring the cut surface was parallel to the 0-0 axis of this coordinate system. Another random number was chosen, and the tissue was sliced again along this second number. The tissues were then sectioned using a microtome into slices of 5- and 20-microns thickness. These sections were placed on slides and subsequently underwent staining using Hematoxylin and Eosin (H&E) method, which involved placing the slides in special staining baskets for coloration.
Several key measurements were taken from the ovarian samples. The total volume of the ovary, as well as the volumes of the cortex and medulla, were calculated using the Cavalieri method [34]. In addition, the volume of the corpus luteum was also determined using the same method. The volume of the oocyte and its nucleus in various types of ovarian follicles were measured using the nucleator method. Lastly, the number of different types of ovarian follicles was determined using the disector method [35]. This method is a stereological technique that provides unbiased estimates of cell numbers within a defined volume.
The data obtained from the study were rigorously analyzed using SPSS 23.0 software. The statistical tests employed included one-way ANOVA and the Tukey Test. These tests were used to compare the means of the different groups and identify any significant differences. A p-value of less than 0.05 was considered statistically significant.

RESULTS AND DISCUSSION
The study revealed a significant reduction in the mean total volume of the ovary, the volume of the cortex, and the volume of the corpus luteum in mice treated with AuNPs compared to the control group (p<0.01). Interestingly, when comparing the simultaneous treatment group (those treated with both AuNPs and green tea extract) to the group treated solely with AuNPs, there was a notable increase in the average total ovarian volume, cortical volume, and corpus luteum volume (p<0.01). This indicates that the combination of AuNPs and green tea extract may counteract some of the effects of AuNPs alone, leading to an enhancement in these specific ovarian parameters (Fig. 1). 
{insert Fig. 1}
The study observed a significant decrease in the average number of primordial, primary, secondary, and tertiary follicles in the group treated with AuNPs when compared to the control group (p<0.01). However, in the group that received simultaneous treatment with AuNPs and green tea extract, this reduction in follicle numbers was significantly compensated (p<0.01). The follicle count in this group was comparable to that of the control group, indicating that the combined treatment may counteract the inhibitory effects of AuNPs on follicular development.
Furthermore, the group treated with green tea extract alone exhibited a significant increase in the average number of primordial, primary, secondary, and tertiary follicles compared to the control group (p<0.01). This suggests that green tea extract may have a stimulatory effect on follicular development (Fig. 2).
{insert Fig. 2}
The mean oocyte volume in primordial, primary, secondary, and tertiary follicles in the group treated with AuNPs was significantly reduced compared to all other groups (p<0.01). In the group that received simultaneous treatment with AuNPs and green tea extract, this reduction was significantly compensated, bringing the mean oocyte volume back to the level observed in the control group (p<0.01)). Furthermore, the mean oocyte volume of the aforementioned types of follicles increased significantly in the group treated solely with green tea extract, compared to the control group (p<0.01) (Table 1).
{insert Table 1}
The mean volume of the oocyte nucleus in primordial, primary, and secondary follicles in the group treated with AuNPs was significantly reduced compared to the control group (p<0.01). In contrast, a significant increase was observed in the average volume of oocytes nucleus in the green tea extract group compared to the control group (p<0.01). In the group that received simultaneous treatment with AuNPs and green tea extract, the decrease in the volume of the oocyte nucleus in primordial, primary, and secondary follicles observed in the AuNPs group was significantly compensated, returning to the levels observed in the control group (p<0.01) (Table 2).
{insert Table 2}
The results of this study provide compelling evidence that green tea extract can help mitigate AuNPs-induced damage to ovarian tissue in adult female SD rats. Specifically, the data demonstrates that administering green tea extract concurrently with AuNPs significantly preserves ovarian morphological parameters including total ovarian volume, cortical volume, medullary volume, and corpus luteum volume compared to treatment with AuNPs alone (Fig. 1). These findings align with previous research which has established the antioxidant and protective capacities of green tea extract against nanoparticle toxicity. For example, a study found that green tea extract alleviated testicular tissue impairment triggered by silver nanoparticles in Wistar rats, indicated by improved sperm parameters [18]. The researchers attributed these beneficial impacts to the free radical scavenging abilities of green tea extract catechins including EGCG.
In the current study, morphological analysis further revealed a drastic decline in the number of ovarian follicles across all developmental stages, primordial, primary, secondary and graafian upon AuNPs treatment relative to control animals (Fig. 2). This aligns with earlier reports demonstrating the suppressive effect of nanoparticles like silver and titanium dioxide on folliculogenesis [25,28,31]. However, green tea extract co-administration was notably effective in recovering follicular abundance, restoring it to levels comparable to the control group. Green tea extract supplementation alone also elicited significant improvement beyond the control baseline. The oocyte and nuclear volumes within follicles exhibited similar patterns among groups (Table 1 and 2).
These enhancements in morphological and quantitative traits could be attributed to the antioxidant and free radical quenching properties of green tea extract polyphenols like EGCG [32]. By mitigating nanoparticle-mediated oxidative damage, green tea enables preservation of ovarian tissue architecture. The stimulatory effect of green tea extract on follicular development observed here corroborates past studies which have suggested green tea supports female reproductive parameters through multiple mechanisms, enhancing ovarian angiogenesis, blocking pathways triggering follicular atresia, stimulating granulosa cell proliferation etc. [15].
Thus, this study provides robust evidence that green tea extract holds immense promise as a protective agent against ovarian perturbations induced by AuNPs specifically, and potentially nanomaterials in general. The ability of green tea extract supplementation to rescue morphological, histological and quantitative ovarian traits from AuNPs-mediated toxicity bears noteworthy clinical relevance. These findings merit further investigation to enable translational applications promoting female reproductive health against increasing nanoparticle exposures.

CONCLUSIONS
This study provides compelling evidence supporting the promise of green tea extract as an ovario-protective agent capable of mitigating AuNPs-induced toxicity. Co-supplementation with green tea extract was notably effective in recovering morphological traits like ovarian, cortical, medullary and corpus luteum volumes along with quantitative parameters such as follicle counts and oocyte/nuclear volumes compared to standalone AuNPs treatment. The antioxidative and free radical scavenging properties of green tea extract, mediated by bioactive polyphenols, seem to promote ovarian health by alleviating nanoparticle-triggered oxidative damage. These impacts could mitigate downstream effects including impaired folliculogenesis, suppressed steroidogenesis and reduced fertility. The present findings align with and substantially build upon previous preliminary research demonstrating reproductive benefits of green tea extract.
While further studies are warranted exploring translational applications, this investigation substantiates the promising potential of green tea extract to promote female reproductive health amidst increasing environmental nanoparticle exposures. From a clinical perspective, incorporating green tea extract as a dietary supplement could provide a safe, economical and readily accessible solution to attenuate ovarian perturbations induced by inadvertent nanoparticle toxicities. Going forward, high-throughput transcriptomic and metabolomic profiling could uncover the precise biomolecular mechanisms governing these protective synergies between green tea extract and gold nanoparticles within ovarian microenvironments. 

CONFLICTS OF INTEREST
The authors affirm that no conflict of interest exists concerning the publication of this manuscript.
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List of Tables:
Table 1. Comparison of the mean volume of oocytes (µm3) in primordial, primary, secondary and tertiary follicles across different SD rat groups, 28 days post-treatment with AuNPs (200 mg/kg/day) and green tea extract (10 ml/kg/day)
Table 2. Comparison of the mean volume of oocytes nucleus (µm3) in primordial, primary, secondary and tertiary follicles across different SD rat groups, 28 days post-treatment with AuNPs (200 mg/kg/day) and green tea extract (10 ml/kg/day)







 
Table 1. Comparison of the mean volume of oocytes (µm3) in primordial, primary, secondary and tertiary follicles across different SD rat groups, 28 days post-treatment with AuNPs (200 mg/kg/day) and green tea extract (10 ml/kg/day)
Groups	Primordial	Primary	Secondary	Tertiary
Control	1771.23 ± 145.11a	62529.48 ± 481.27a	92725.13 ± 3600.93a	187217.98 ± 8311.55a
AuNPs	1375.47 ± 85.18b	4346.04 ± 340.48b	72990.07 ± 4705.97b	137169.04 ± 37088.95b
Green tea extract	1757.28 ± 84.06a	6010.98 ± 631.88a	85876.88 ± 5991.07ab	167242.42 ± 7852.99ab
Combined	2098.26 ± 279.90c	7893.07 ± 1707.64c	133621.92 ± 22883.60c	227244.33 ± 14744.00c



















Table 2. Comparison of the mean volume of oocytes nucleus (µm3) in primordial, primary, secondary and tertiary follicles across different SD rat groups, 28 days post-treatment with AuNPs (200 mg/kg/day) and green tea extract (10 ml/kg/day)
Groups	Primordial	Primary	Secondary	Tertiary
Control	433.11 ± 29.22a	892.95 ± 40.30a	4366.68 ± 239.05a	6429.93 ± 352.98a
AuNPs	301.14 ± 3.76b	753.01 ± 27.98b	3568.52 ± 151.22b	5534.63 ± 279.96b
Green tea extract	422.01 ± 17.92a	865.40 ± 78.79a	4065.27 ± 426.87a	7242.24 ± 411.54c
Combined	515.49 ± 36.78c	1143.27 ± 100.56c	6204.59 ± 679.83c	9797.65 ± 746.62d









 
List of Figures:
Fig 1. Comparison of the mean volumes of the total ovary, cortex, medulla, and corpus luteum (µm3) across different SD rat groups, 28 days post-treatment with AuNPs (200 mg/kg/day) and green tea extract (10 ml/kg/day)
Fig. 2. Comparison of the mean number of primordial, primary, secondary and tertiary follicles across different SD rat groups, 28 days post-treatment with AuNPs (200 mg/kg/day) and green tea extract (10 ml/kg/day)






 
 
Fig. 1. Comparison of the mean volumes (µm3) of the total ovary, cortex, medulla, and corpus luteum across different SD rat groups, 28 days post-treatment with AuNPs (200 mg/kg/day) and green tea extract (10 ml/kg/day)
	Ovary	Cortex	Medulla	Corpus luteum
Control	2.147 ± 0.1615	1.729 ± 0.095	0.3895 ± 0.0665	0.456 ± 0.095
AuNPs	1.3205 ± 0.1425	0.9975 ± 0.1045	0.2945 ± 0.0665	0.0855 ± 0.038
Green tea extract	2.242 ± 0.418	1.7765 ± 0.247	0.4465 ± 0.19	0.456 ± 0.1425
Combined	2.831 ± 0.437	2.261 ± 0.4085	0.513 ± 0.0475	0.665 ± 0.095














 
Fig. 2. Comparison of the mean number of primordial, primary, secondary and tertiary follicles across different SD rat groups, 28 days post-treatment with AuNPs (200 mg/kg/day) and green tea extract (10 ml/kg/day)
	Primordial	Primary	Secondary	Tertiary
Control	1746.974 ± 408.4145	1191.1005 ± 262.276	898.282 ± 109.0695	90.3735 ± 24.6335
AuNPs	537.472 ± 125.495	323.608 ± 77.7005	119.5195 ± 50.2265	23.6075 ± 2.4795
Green tea extract	2802.9085 ± 731.0345	1756.075 ± 432.25	1093.2505 ± 124.963	82.612 ± 9.557
Combined	5250.099 ± 1230.9435	3000.138 ± 459.7145	2011.5015 ± 316.7205	195.8805 ± 33.2975






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