{"id":61238,"date":"2024-09-30T11:44:15","date_gmt":"2024-09-30T11:44:15","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=61238"},"modified":"2024-10-09T17:54:45","modified_gmt":"2024-10-09T17:54:45","slug":"neem-leaves-extract-reduces-sex-steroids-and-gonadal-function-in-female-wistar-albino-rats","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/neem-leaves-extract-reduces-sex-steroids-and-gonadal-function-in-female-wistar-albino-rats\/","title":{"rendered":"Neem Leaves Extract Reduces Sex Steroids and Gonadal Function in Female Wistar Albino Rats"},"content":{"rendered":"\n

 <\/em><\/strong>Introduction\n<\/strong><\/p>\n\n\n\n

One of the most serious global problems is stray dogs overpopulation, which has an adverse impact on public health, the environment, and communities, various zoonoses have developed and become endemic as a result of this problem 1<\/sup>. Thus, it is important to search for a new effective method with low side effects to solve this problem, such as medicinal plants with antifertility effects. Moreover, the human population explosion is considered a major cause of human beings’ suffering and environmental squalor all over the world 2<\/sup>. Thus, it is important to search for a new effective method with low side effects to solve this problem. Synthetic contraceptives possess an effective role in solving such problems however, they are not safe as they may cause allergies or heart attack3<\/sup>. <\/p>\n\n\n\n

Since ancient times, herbs have been reported for their useful remedial influences against various disease conditions owing to their pharmacological belongings4<\/sup>. Therefore, plants have provided humans with new medicinal solutions for thousands of years, acting as a basis for traditional medical systems worldwide5<\/sup>. Herbs could be used as an alternative to drugs of synthetic origin and they have gained a recent popularity among developing countries due to their affordability, availability and abridged side\/toxicity influences 6,7<\/sup>. A variety of medicinal plants that were previously proved to have antifertility action could be industrialized into contraceptives for both females and males8<\/sup>. They can cause estrous cycle disruption, anti-estrogenic effects, anti-implantation influence, or even abortion9<\/sup>. <\/p>\n\n\n\n

One of the most significant medicinal plants found in Asia and Africa is neem (Azadirachta indica<\/em>), which is rich in proteins and trace minerals, has Anti-inflammatory and Antioxidant effects, and can be used to treat a variety of animal parasites, bacteria, and viruses10<\/sup>. Additionally, fresh neem leaves contain polyphenolic flavonoids with antibacterial and antifungal effects, Furthermore, neem seeds contain beneficial components like azadirachtin and gedunin11<\/sup>.  Gbotolorun, Osinubi, Noronha and Okanlawon12<\/sup> detected that the antifertility effects of the neem flower extract on adult female rats, causing disruption of the estrous cycle and a partial blockage in the ovulation. Patil Patil, Shirahatti, VB, Ramu and Prasad13<\/sup> detected that the consumption of A. indica<\/em> does not cause any harm to the entire reproductive system, nevertheless, has the ability to be used as a temporary or reversible contraceptive. This study aimed to investigate the possible anti-fertility potential of dietary neem leaves’ extract in female Wister albino rats as a model to be further applied to stray dogs.                                                                                                                                 <\/p>\n\n\n\n

Materials and Methods <\/strong><\/p>\n\n\n\n

Plant Extract <\/strong><\/p>\n\n\n\n

Creation of Crude Aqueous Neem Extract <\/strong><\/p>\n\n\n\n

According to Mamoon-ur-Rashid,\nAbdullah and Hussain. 14<\/sup>, the Azadirachta indica<\/em> tree\nleaves were dried, then grinding and homogenized with distilled water in an electric blender, and triple-folded\ngauze was used to filtrate the homogenate. Before usage, a rotary vacuum\nevaporator was used to evaporate\nthe solvent, then 70% dilation of the extracts was prepared.<\/p>\n\n\n\n

Experimental Animals<\/strong><\/p>\n\n\n\n

A total\nof 24 mature female rats (5.5\u20136 months; 180\u2013200 g) of Wistar strain were\ncastoff in this study. Animals were acquired from Laboratory Animal House,\nFaculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. They\nwere housed in polyethylene cages; four females per cage at room temperature (25\u00b0C \u00b1\n1\u00b0C) and natural daylight cycles. Water and feed were offered ad libitum. Rats\nwere kept 1 week to adapt.\nThe experimental producers adhered to the ethical rules for the utilization of\nanimals in laboratory settings at the Faculty of Veterinary Medicine, Suez\nCanal University, Egypt (SCU-VET 2024032).<\/p>\n\n\n\n

Reproductive Procedures<\/strong><\/p>\n\n\n\n

After 1\nweek of adaptation, daily cytological inspection of vaginal smears was done to\ndetect the estrous cyclicity progression and regularity. Females that revealed two\nsuccessive regular cycles were designated for the current study and others were\nexcluded. <\/p>\n\n\n\n

Design of the Experiment <\/strong><\/p>\n\n\n\n

Twenty-four\nregular cyclic females were split\nequally into two groups; G (I): control group (n = 12) was fed a basal diet misted\nwith 53 mL distilled water and G (II): neem extract group (n = 12) that was fed\nbasal diet misted with 3 mL neem leaves extract mixed with 50 mL distilled\nwater with a dose 10 mL neem leaves extract \/ kg diet. A total of 200 g diet was offered \/ cage.\nThe experimental diet was offered daily for 30 days. <\/p>\n\n\n\n

Estrous Regularity<\/strong><\/p>\n\n\n\n

After 15\ndays from the start of the\nexperiment, vaginal smears were smeared from individual rats every 12 hours to detect\nthe average duration\/hours for each estrous cycle phase for two consecutive\ncycles. <\/p>\n\n\n\n

Sampling<\/strong><\/p>\n\n\n\n

At the\nend of 30 days of\ntreatment, 3 females representing each stage of the cycle were sacrificed.\nBlood was drawn from retro-orbital venous vessels allowed to clot then\ncentrifugated at 3000 rpm to obtain serum. Serum was kept at -70o<\/sup>C.  The uterus and ovaries of each female were\ndissected and weighed. <\/p>\n\n\n\n

Feed Intake and Relative Sex Organs Weights<\/strong><\/p>\n\n\n\n

Final body\nweights were recorded. The feed intake for rat\neach experimental animal was calculated.  The food remaining was subtracted from the offered food then the\nobtained value was divided by the number of rats per cage. The dissected ovaries\nand uteri were weighed, and their relative weights were obtained as follows (organ weight\/body\nweightX100). <\/p>\n\n\n\n

Sex Steroids Levels<\/strong><\/p>\n\n\n\n

Serum\nlevels of 17-\u03b2 estradiol of females in the follicular phase of the cycle (proestrus and\nestrus phases) and serum progesterone levels in the luteal phase of the cycle (metestrus and\ndiestrus) were determined using Kamiya Biomedical Company, ELISA kits (USA).\nBoth analyses were performed according to the enclosed pamphlet instructions.<\/p>\n\n\n\n

Histopathology<\/strong><\/p>\n\n\n\n

Part of the\ndissected uteri and ovaries were put in a 10% formalin solution. Afterward, they were\nimmersed into paraffin wax and stained by hematoxylin and eosin15<\/sup>.<\/p>\n\n\n\n

Ovarian Caspase-3 Expression <\/strong><\/p>\n\n\n\n

One ovary\/ animal was kept at -80o<\/sup>C until RNA extraction has proceeded. The frozen ovaries were subjected to RNA extraction using a total RNA extraction kit (QIAGEN, Maryland, USA), as mentioned in pamphlet of the manufacturer. The complementary DNA was synthesized using (Thermo Fisher Scientific Inc., Lithuania) kit according to manufacturer\u2019s instruction. Gene expression of ovarian caspase-3 against the housekeeping gene \u03b2-actin following He, Sun and Huang 16 <\/sup>primer sequence and methodology.  Primer for caspase-3 was Forward: 5\u2032-GTGGAACTGACGATGATATGGC-3\u2032 and reverse:  5\u2032-CGCAAAGTGACTGGATGAACC-3\u2032. Primers for \u03b2-actin were Forward: 5\u2032-AAGATCCTGACCGAGCGTGG-3\u2032 and reverse: 5\u2032-CAGCACTGTGTTG GCATAGAGG-3\u2032. Fold change 2-\u0394\u0394Ct was implemented to estimate the levels of gene expression of caspase-3. <\/p>\n\n\n\n

 Statistical Analysis<\/strong><\/p>\n\n\n\n

The\nresults obtained in the present work were calculated by student t-test. Then\nthe data were expressed as a mean \u00b1 standard error (mean \u00b1 SE), where p \u2264 0.05\nis considered statistically significant. All of the analyses were carried out\nusing the R programming language17<\/sup><\/strong>.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

The Shapiro-Wilk test for univariate normality showed that the data were normally distributed (p>0.05).<\/p>\n\n\n\n

Estrous Regularity<\/strong><\/p>\n\n\n\n

Experimental rats had significantly (p<0.05) longer\ndiestrus phases in the\nneem leaves extract administered group compared to controls.<\/strong> While these rats had\nno significant differences in the duration of proestrus, estrous, and metestrus\nphases (Table 1).<\/p>\n\n\n\n

 Table 1: Effect of neem leaves extract on estrous cycle duration of female albino rats.<\/strong><\/p>\n\n\n\n\n\n\n\n
\n

Group<\/strong><\/p>\n<\/td>\n

\n

Proestrus (h)<\/strong><\/p>\n<\/td>\n

\n

Estrous (h)<\/strong><\/p>\n<\/td>\n

\n

Metestrus (h)<\/strong><\/p>\n<\/td>\n

\n

Diestrus (h)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Neem extract treated group<\/p>\n<\/td>\n

\n

 0.93<\/p>\n<\/td>\n

\n

 0.95<\/p>\n<\/td>\n

\n

 0.50<\/p>\n<\/td>\n

\n

 3.43<\/p>\n<\/td>\n<\/tr>\n

\n

Control group<\/p>\n<\/td>\n

\n

11 0.52<\/p>\n<\/td>\n

\n

19.67 1.20<\/p>\n<\/td>\n

\n

10.33 0.33<\/p>\n<\/td>\n

\n

63.67 3.88<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Data was expressed as a mean \u00b1 standard\nerror (mean \u00b1 SE) then data was analyzed by student t-test using analyses carried out using the R\nprogramming language. NS means that neem leaves extract group was non significantly varied (p0.05) than the control group.* means there was a significant difference\nat p<0.05. Control group (n = 12) was fed a basal diet misted with 53 mL distilled water. Neem\nextract group (n = 12) was\nfed a basal diet misted with 3 mL neem leaves extract mixed with 50 mL distilled\nwater with a dose 10 mL neem leaves extract \/ kg diet.<\/p>\n\n\n\n

Feed Intake and Relative Sex Organs Weights<\/strong><\/p>\n\n\n\n

Female rats treated with neem extract had a mean rat feed intake of 24.42 (g\/day), compared to 24.23 (g\/day) in the control group. The level of rat feed intake in treated female rats is approximately the same and non-significantly (p>0.05) altered as matched to controls (Figure 1). Concerning ovarian and uterine relative weights, there were non-significant (p>0.05) differences observed between neem leaves extract group and the control one either in follicular or luteal phases of the cycle (Table 2).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Effect of neem leaves extract on rats\u2019 feed intake. Data is expressed as a mean \u00b1 standard error (mean \u00b1 SE) then data was analyzed by student t-test using analyses carried out using the R programming language.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 2: Influence of neem leaves extract on rats\u2019 relative ovarian and uterine weights.<\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n<\/td>\n

\n

Ovarian relative weight (%)<\/strong><\/p>\n<\/td>\n

\n

Uterine relative weight (%)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Follicular phase<\/strong><\/p>\n<\/td>\n

\n

Luteal phase<\/strong><\/p>\n<\/td>\n

\n

Follicular phase<\/strong><\/p>\n<\/td>\n

\n

Luteal phase<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Neem Extract treated group<\/p>\n<\/td>\n

\n

 0.003<\/p>\n<\/td>\n

\n

 0.004<\/p>\n<\/td>\n

\n

 0.018<\/p>\n<\/td>\n

\n

 0.011<\/p>\n<\/td>\n<\/tr>\n

\n

Control group<\/p>\n<\/td>\n

\n

0.<\/em>066 0 0.02<\/p>\n<\/td>\n

\n

 0.004<\/p>\n<\/td>\n

\n

0.518 0.026<\/p>\n<\/td>\n

\n

0.485 0.006<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Data is expressed as a mean \u00b1 standard error (mean \u00b1 SE) then data was analyzed by student t-test using analyses were carried out using the R programming language NS means that Neem leaves extract group was non significantly varied (p>0.05) than control group.  Control group (n = 12) was fed a basal diet misted with 53 mL distilled water. Neem extract group (n = 12) was fed a basal diet misted with 3 mL neem leaves extract mixed with 50 mL distilled water with a dose 10 mL Neem leaves extract \/ kg diet.<\/p>\n\n\n\n

Sex Steroids Levels<\/strong><\/p>\n\n\n\n

Female\nrats treated with neem leaves extract had a mean serum estradiol content of\n16.45 (pg\/mL), compared to 25.26 (pg\/mL) in the control group. The level of\nestradiol in treated female rats is approximately 34.88% lower and significant\n(p<0.05) as compared to controls (Table 3).<\/p>\n\n\n\n

Female rats treated with neem leaves extract had a mean serum progesterone content of 7.83 (ng\/mL), compared to 12.70 (ng\/mL) in the control females. In comparison to controls, the level of progesterone in treated female rats is approximately 38.35% lower and significant (p<0.05) (Table 2).<\/p>\n\n\n\n

Table 3: Effect of neem leaves extract on serum levels of sex hormones in female albino rats.<\/strong><\/p>\n\n\n\n\n\n\n\n
\n

Group<\/p>\n<\/td>\n

\n

Serum estradiol (pg\/mL)<\/p>\n<\/td>\n

\n

Serum progesterone (ng\/mL)<\/p>\n<\/td>\n<\/tr>\n

\n

Neem Extract treated group<\/p>\n<\/td>\n

\n

1.39<\/p>\n<\/td>\n

\n

 1.09<\/p>\n<\/td>\n<\/tr>\n

\n

Control group<\/p>\n<\/td>\n

\n

25.26 2.30<\/p>\n<\/td>\n

\n

12.70 1.43<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Data is expressed as a mean \u00b1 standard\nerror (mean \u00b1 SE) then data was analyzed by student t-test using analyses carried out using the R\nprogramming language. * means there was significant difference at p<0.05. Control\ngroup (n = 12) was fed a\nbasal diet misted with 53 mL of distilled water. Neem extract group (n = 12) was fed a basal diet\nmisted with 3 mL neem leaves extract mixed with 50 mL distilled water with a\ndose 10 mL of neem leaves extract \/ kg diet.<\/p>\n\n\n\n

Histopathology<\/strong><\/strong><\/p>\n\n\n\n

Microscopically, the ovary of control rats in estrus (CE) was covered externally by a single cuboidal cells layer, germinal epithelium. Additionally, a thick layer of connective tissue rich in fibroblasts; tunica albuginea, was observed beneath the covering epithelium. The cortical region revealed various follicular stages along with stromal cells interspersed in between. The most numerous types of ovarian follicles were primordial follicles that mainly formed by a primary oocyte covered by a monolayer of squamous granulosa cells. Within the ovarian cortical tissue, the primary follicles were recorded. It was larger in size than the primordial one. It was formed by a larger primary oocyte covered by a monolayer of cuboidal granulosa cells. Late and early stages of secondary follicles were noted, whereas the largest primary oocytes were bounded by numerous layers of granulosa cells that are polyhedral in shape with the various fluid-filled places among the granulosa cells that are covered outwardly by theca cells. Those multiple spaces were merged with each other founding a single big antrum (Figure 3). On the opposite, the ovarian tissue of females fed neem extract in estrus (NE) displayed the same histoarchitecture as that of CE with some differences; the cortical tissue was composed mainly of  dense irregular connective tissue rich in fibroblasts and other connective tissue cells that surrounds the ovarian follicles, the follicular developmental stages were declined than that of CE, and finally, great numbers of blood capillaries were evident within the ovarian medulla (Figure 3).<\/p>\n\n\n\n

Uterine tissue sections that stained with H&E of CE showed\ninner folded endometrial mucosa (simple columnal epithelium and lamina\npropria-submucosa contained uterine glands), myometrial middle (outer\nlongitudinal bundles and inner circular of smooth muscles along with stratum\nvascular separating) and outermost perimetrium (contained simple squamous\nepithelium surrounding a connective tissue layer) (Figure 3). <\/strong>The uterus\nof NE displayed the same histological structures as CE, meanwhile the most\nprominent uterine findings in NE were the uterine epithelial stratification,\nfew or no uterine glands were recorded in the lamina propria-submucosa and\nproliferation of blood capillaries was observed in perimetrium (Figure 3).<\/p>\n\n\n\n

During the luteal phase, the ovarian sections of control females during metestrus (CM) demonstrated highly active and well-developed corpora lutea with abundant granulosa lutein cells enclosed by a well-vascularized connective tissue capsule (Figure 4).However, the ovarian tissue of the neem group (NM was intermingled and characterized by rudimentary ovarian follicles, a huge amount of connective tissue with connective tissue cells and the proliferation of blood capillaries (Figure 4). <\/strong>It was interesting to note that the ovary of both CM and NM groups was covered externally by a single layer of flat cells, germinal epithelium.<\/p>\n\n\n\n

The uterine tissue sections of CM\nand NM showed the same histo-architecture as follows; inner endometrium (simple\ncolumnal epithelium and lamina propria-submucosa contained uterine glands),\nmiddle myometrial layer (longitudinal bundles and inner circular outer of\nsmooth muscles along with stratum vascular separating) and outer perimetrium\n(contained simple squamous epithelium surrounding a connective tissue layer) (Figure\n4).<\/p>\n\n\n\n

Caspase-3-fold change expression<\/strong><\/p>\n\n\n\n

Figure (2) revealed a significant increase (p<0.05) in the fold change mRNA of ovarian caspase-3 in the neem leaves extract group as compared to control during the luteal and follicular phases of the estrous cycle. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Effect of neem leaves extract on fold change expression of ovarian caspase-3 in female albino rats.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Representative photomicrographs of H&E-stained ovarian and uterine tissues sections during estrus phase of follicular stage of the estrous cycle.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 4: Representative photomicrographs of H&E-stained ovarian and uterine tissues sections during metestrus phase of luteal stage of the estrus cycle. <\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion <\/strong><\/p>\n\n\n\n

Neem (Azadirachta\nindica<\/em>) is recognized as a highly potent natural contraceptive 18<\/sup>. Employing A. indica as a contraceptive offers several benefits due to\nits safety, effectiveness, and reversibility in both genders 19<\/sup>. The\ncontraceptive properties of A. indica are linked to its capacity to interfere\nwith various aspects of the female reproductive system, such as altering\nhormonal balance 20<\/sup>, causing irregularities in the estrous cycle 21<\/sup>, and inducing apoptosis in ovarian cells 7<\/sup>, while having no adversative properties on productive performance 22-24<\/sup>. <\/p>\n\n\n\n

The usage of chemical or synthetic\ncontraceptives could also alter the hormonal balance and reproductive cycle that impede ovarian\nand uterine functions 25<\/sup>. Among these effects; are the suppression of\nfolliculogenesis, follicle\n development and suppression of\novulation. Moreover, most of them are hormones that interact with hypothalamic\nendocrine neurons to suppress pituitary gonadotropins 26<\/sup>. The usage\nof these contraceptives could produce myriads of side effects such as hypertension,\nbreast cancer susceptibility, dyslipidemia and susceptibility to thrombosis 27<\/sup>\nand cardiovascular diseases 28<\/sup> that made herbal contraceptives a\nsafe  alternative.<\/p>\n\n\n\n

Our treated rats\nshowed that A. indica extract significantly prolongs the diestrus phase without\naltering the durations of the proestrus, estrus, and metestrus phases. Such a result aligned with\nthat of Mamoon-ur-Rashid, Abdullah and Hussain 12<\/sup> who declared\nprolonged diestrus in female rats that were given 1 g\/kg body weight of alcoholic neem\nflower extract with a total 80% estrous irregularity percentage. Auta and\nHassan 29<\/sup> found that neem extract administration at doses 5, 50 and\n100 mg\/kg neem wood aqueous extract for 20\ndays in mice\nproduced a significant prolongation in the diestrus phase in a dose dependent pattern. Sitasiwi, Isdadiyanto and Mardiati 19<\/sup>, also reported that\nethanolic A. indica leaves at levels 8.4, 11.2, and 14\nmg\/mice\/day for 21 days can disrupt the\nregularity of the estrous cycle and prolong diestrus in Swiss Webster\nmice. Despite the\nsignificant physiological disruptions within the estrous cycle, A. indica<\/em> extract\ndid not significantly affect mean daily feed intake, or the relative weights of\nthe ovaries and uterus as shown in herein study and in parallel with El-Zaiat,\nElshafie, Al-Marzooqi and Dughaishi 30<\/sup> as well as Biswas,\nChattopadhyay, Banerjee and Bandyopadhyay 31<\/sup>. This implies that the\ncontraceptive effects of A. indica<\/em> extract are not due to changes in\noverall nutrition, the\nweight of reproductive organs or body conditions 19,21,32<\/sup>. So, the extension of the diestrus phase in neem-treated rats suggests that A.\nindica extract disrupts the hormonal feedback mechanisms essential for the\nprogression of the estrous cycle. Where the estrous cyclicity is completely\ngoverned by neuroendocrine feedback that is completely controlled by anterior\npituitary gonadotropins secretion and gonadal steroids production 33-35<\/sup>\nthat seemed to be reduced in the current study (noticed by 17-\u03b2 estradiol and\nprogesterone reductions).  <\/p>\n\n\n\n

Normally, an\nincrease in 17-\u03b2 estradiol signals the end of the diestrus phase and the start\nof a new cycle 36<\/sup>. However, in neem-treated rats, 17-\u03b2 estradiol\nlevels were about 34.88% lesser in females those in the control group during the follicular\nphase. The former result was aligning with observations mentioned by Sitasiwi,\nIsdadiyanto and Mardiati who reported that ethanolic A. indica in leaves 8.4, 11.2, and 14\nmg\/mice\/day for 21 days can reduce 17-\u03b2\nestradiol in mice 19<\/sup>. Also, Shaikh,\nNaqvi and Khani 37<\/sup> demonstrated that neem oil administration to\nfemale albino rats at doses 0.6 and 1.2 mL\/animal significantly reduced 17-\u03b2 estradiol in the\nblood. Moreover, Tripathi, Shrivastav and Chaube 38<\/sup> detected lower\n17-\u03b2 estradiol in follicular lysate after administration 50\nmg\/day aqueous neem extract to immature female rats for 10 days. This reduction suggests that the hormonal signal, needed to end diestrus\nand trigger follicular development and maturation (proestrus) 39<\/sup>, is\nimpaired. As a result, the hormonal threshold for transitioning from diestrus\nmight not be reached, leading to an extended diestrus phase. These decreased\n17-\u03b2 estradiol levels imply that A. indica extract exhibits strong\nanti-estrogenic effects19<\/sup>.<\/p>\n\n\n\n

Our findings show\nthat serum progesterone levels were significantly reduced, approximately 38.35%\nlower than in controls, which\nin line with the study by Moravati, Mahmoudi, Ghazi-Khansari, Aria and Jabbari  40<\/sup> who found that\ngavage of neem methanolic extract at a dose of 15 mg\/ kg for 6 days significantly abridged blood\nprogesterone level in female rats that support the strong anti-progestational effects exerted by A. indica extract.\nMoreover, Physiologically, progesterone plays a crucial role in maintaining the\ndiestrus phase, with its decline signalling the end of this phase 39<\/sup>.\nProgesterone upregulation signals neuroendocrine reflex through the\nhypothalamus to promote pituitary gonadotropin production to establish a new cycle. The reduction\nof progesterone level in the herein study led to prolongation of diestrus due\nto delay of its feedback on hypothalamic releasing hormones that delay pituitary\ngonadotropins secretion thus hindering initiation of new cycle 41<\/sup>.\nThis interference might involve altering progesterone receptor sensitivity,\nacting as a progesterone receptor blocker, or disrupting the signalling\npathways that process these hormone levels, resulting in a delayed transition\ndespite low progesterone 42<\/sup>. The signalling pathways that process\nprogesterone involve dimerization to receptors where there are different\nprogesterone receptor\n(PR) subtypes related to reproductive function (PR-A, PR-B and PR-C) as well as\nG-protein coupled membrane receptors. \nPR-A limits transcription of target genes referring to the\nactive inhibitory domain that prohibits further translation into new proteins.\nHowever, PR-B receptors stimulate DNA\ntranscription and further translation to new proteins specifically nuclear receptor\ncoactivator that has a close association with maintenance of wet uterine weight\n43<\/sup>. PR-C lacks DNA binding\naffinity as PR-A or PR-B but it can dimerize receptors as a heterodimer that is not as effective\nas a homodimer43<\/sup>. The G protein coupled PR exerts its regularity\nfunction in ovaries and hypothalamus via inhibition of cAMP 44<\/sup>. The\nablation of PR function by neem could contribute to altering of neuroendocrine\nregulation exerted by such receptors in reproductive cycle regulation. <\/p>\n\n\n\n

Beyond their role\nin the progression of the estrous cycle, 17-\u03b2 estradiol and progesterone have\nprotective, anti-apoptotic effects on ovarian cells, supporting the proper\ndevelopment and maintenance of follicles and the corpus luteum 45<\/sup>.\nWhen these hormone levels decline, ovarian caspase-3 activity increases, as\ndemonstrated in our results. The increase in ovarian caspase-3 expression may\nbe due to neem suppression to catalase activity that promotes H2<\/sub>O2<\/sub>\ninduction to BAX and P53 that hasten caspase-3 expression46<\/sup>.\nCaspase-3 is a key executioner of apoptosis 47<\/sup>. Its activation\ntriggers the cleavage of cellular proteins, leading to programmed cell death.\nThis process constitutes a crucial part in the follicular atresia of\nnon-dominant follicles during the follicular phase and the relapse of the\ncorpus luteum during the luteal phase if pregnancy does not occur 48<\/sup>.\nConsequently, increased caspase-3 activity leads to heightened apoptosis,\ndisrupted ovarian function, and increased follicular atresia. This results in\nfewer follicles maturing and ovulating, accompanied by increased connective\ntissue in the ovary compared to control groups, as observed in the present\nhistological examination. These findings are consistent with studies by Akpantah,\nEkong, Obeten, Akpaso and Ekanem 49<\/sup> in female rats and Swamy and\nMohan 50<\/sup> in freshwater fish.<\/p>\n\n\n\n

Additionally, insufficient endometrial\nproliferation and glandular development are consequences of reduced levels of\n17-\u03b2 estradiol in A. indica-treated rats 51<\/sup>. This explains the\nobserved epithelial stratification and the scarcity of uterine glands in our\nhistological sections, which\nalign with the finding of Auta and Hassan 29<\/sup>  who found\nthat neem extract administration doses of 5,\n50, and 100 mg\/kg neem wood extract for 20 days to mice produced uterine\nepithelial stratification especially in 50 mg and 100 mg doses . Furthermore, the low progesterone levels in these treated rats impair\nsecretory functions, leading to diminished glandular activity and compromised\nstructural integrity of the endometrium 29<\/sup>. The absence of proper\nglandular structures in the endometrium reflects impaired uterine preparation\nfor potential implantation, which is consistent with the contraceptive\nproperties of neem extract 29,51<\/sup>.<\/p>\n\n\n\n

Conclusion  <\/strong><\/p>\n\n\n\n

Neem leaves extract produced antifertility potential in female Albino rats through Reducing sex steroids; estrogen in the follicular phase and progesterone in the luteal phase. That led to the prolongation of estrous cycle duration especially the diestrus phase. Moreover, the reduced sex steroids by neem leaves extract promoted an  increment in the ovarian caspase-3 mRNA expression that reflected follicular apoptosis and atrasia.  The previous events provoked histological deteriorations in the ovary such as a reduced number of developing follicles and connective tissue proliferation beside uterine epithelial stratification with diminished uterine glands that reflect their incompetence for ovulation and implantation.  <\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

The authors would like to acknowledge Haneen Mohammed, undergraduate student, Faculty of Veterinary Medicine, Suez Canal University for her help during experimental procedures. <\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors do not have any conflict of interest <\/p>\n\n\n\n

Funding sources<\/strong><\/p>\n\n\n\n

The author(s) received no financial support for the research, authorship, and\/or publication of this article.<\/p>\n\n\n\n

Data Availability<\/strong> Statement<\/strong> <\/p>\n\n\n\n

The manuscript incorporates all datasets produced or examined throughout this research study. <\/p>\n\n\n\n

Ethics Statement<\/strong><\/p>\n\n\n\n

This research did not involve human participants, animal subjects, or any material that requires ethical approval<\/p>\n\n\n\n

Informed Consent Statement<\/strong><\/p>\n\n\n\n

This study did not involve human participants, and therefore, informed consent was not required<\/p>\n\n\n\n

Authors\u2019 Contribution<\/strong><\/p>\n\n\n\n

Conceptualization, E.M.A., Heba M.A. Abdelrazek;<\/p>\n\n\n\n

Methodology, Heba M.A. Abdelrazek, Asem\nA. Awad, Nayrouz A. Attia,\nMohamed R. Saad, Marwa S. Kamel<\/p>\n\n\n\n

Formal analysis, Rana M. Al-awadhi., Heba M.A. Abdelrazek., Eman M.\nAbouelhassan, <\/p>\n\n\n\n

Investigation, Rana M. Al-awadhi., Heba M.A. Abdelrazek., Nayrouz A. Attia.,  Marwa\nS. Kamel.; <\/p>\n\n\n\n

Resources, Rana M. Al-awadhi, Heba\nM.A. Abdelrazek., Mohamed R.\nSaad, Asem A. Awad.; <\/p>\n\n\n\n

Writing- original draft preparation, Nayrouz A. Attia, Mohamed R. Saad., Asem A. Awad; <\/p>\n\n\n\n

Writing-review and Editing, Rana M. Al-awadhi., Heba M.A. Abdelrazek., Eman M. Abouelhassan., Marwa S. Kamel<\/p>\n\n\n\n

All authors have read and agreed to\nthe published version of the manuscript.<\/p>\n\n\n\n

References <\/strong><\/p>\n\n\n\n

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