INTRODUCTION:

The contraception pills used to impede birth approaches that act on the endocrine system. Hormonal contraception consists of one or more synthetic female sex hormones, users of steroid hormone methods subjected to pregnancy rates of less than 1% per year¹. "Combined oral contraceptive pills" were acquired to preclude ovulation by subduing the emancipation of gonadotropins. "Combined hormonal contraceptives", including combined oral contraceptive pills COCPs, distort "follicular development" and counteract ovulation as a principal mechanism of action². The uterus has three main roles in human reproduction: sperm transport, implantation, and upkeep of pregnancy. All of these roles may theoretically be distorted to provoke contraception.

Steroidal contraceptives invoke both myometrial and endometrial histology and distort the functional activity of the uterus³. It is known that both "estrogen and progesterone" upset the programming and multiplication of the epithelial lining of the endometrial layer of the uterus during their menstrual cycle. "Hormonal contraception" dangles upon using exogenous forms of either progesterone or both "estrogen and progesterone", these "exogenous hormones" affecting the "endometrial layer" of the uterus in response to their steady levels of the blood especially⁴.

"Hormonal contraceptive" consumption might weaken bone integrity and escalate the menace of stress fracture by downregulating endogenous estrogen synthesis, a cardinal controller of bone turnover⁵. This study recced bone density in female albino rats taking various kinds of hormonal contraceptives.

MATERIALS AND METHODS:

A total of fifty healthy female Wistar albino rats (age 10-12weeks; weight 200–260gm), were collected from the animal house (College of Veterinary Medicine/University of Mosul). Animals were endorsed in the animal house of Mosul University and were avowed under meticulous settings of temperature (24± 2°C), light-dark periods of 12 hours, and free access to water and a commercial diet. The laboratory guiding principles for biomedical research involving animals were locally optimized and adopted^6,7. The animals were administered a four-week adaptation period after they were located in their new environment.

The animals were divided into five groups, ten rats each. Group I (Control group): Rats receiving no medication and then euthanized for histological examination. Group II (Study group): Rats receiving Yasmin®* "monophasic combined oral contraceptive" tablets (0.03 mg ethinylestradiol and 3mg Drospirenone) once daily through a gastric catheter after being dissolved in one ml distilled water. A dose corresponding to 0.03mg/kg ethinylestradiol and 0.5mg/ kg Drospirenone was given to rats in 5-day cycles (4-day treatment with 1-day break) for eight weeks and then euthanized for histological examination. Group III (Study group): Rats receiving Microgynon pills (Levonorgestrel 0.15 mg/kg+ Ethinylestradiol 0.03mg/kg), orally for 4 days; one day break for 8 weeks. Group IV (Study group): Rats receiving Marvelon pills (Desogestrel 0.15mg/kg+ Ethinylestradiol 0.03mg/kg), orally for 4 days; one day break for 8weeks. Group V (Study group): Rats receiving Depo-Provera injection Progesterone only contraceptive injection (150mg/ml Medroxyprogesterone IM Injection) once weekly through IM injection. A dose corresponding to 3.5mg/ rat was given to rats for eight weeks and then euthanized for histological examination.

The uterus, ovary, cervix, endometrium, mammary glands, and bone samples were cut (1cm³) and instantly fixed in 10% neutral buffered formalin. The tissue was washed in tap water and dehydrated in ascending grades of ethyl alcohols, cleared in xylene, and finally embedded in paraffin wax at 60 C° using (Gestion -ATP7000 tissue processor-Germany). The paraffin was sectioned at 5-6 μm thickness using (Rotary microtome (Gestion ERM 4000 Germany) and stained with Haematoxylin and Eosin according to Suvarna et al, 2013, protocol⁸.

All factors were determined using the "color USB 2.0 digital image camera (Scope Image 9.0- China)" which was supported with image handling software. The software of the "camera was calibrated to all lenses of Microscope-Olympus-CX31" with the aid of a "0.01mm stage micrometer (ESM-11/Japan)".

A "computer package (Sigma Stat V12.0/SYSTAT software)" was used to conduct the histomorphometric analysis. The results were expressed as means±SE (standard error) and were analyzed using Duncan's test with a significant level set on P <0.05 (Systat Software Inc 2016(

RESULTS:

Yasmin group has shown increased density of metaphyseal bone compared to the control group. "Microgynon group" has shown low density of epiphyseal plate cartilage, and low density of trabecular bone compared to control. Marvelon group has shown high density and thickness of articular cartilage, high density of trabecular bone, normal epiphyseal plate cartilage. Depo-Provera group shows thinning and very-low-density articular cartilage with the proliferation of fibrous connective tissue, low density of trabecular bone, and normal epiphyseal plate cartilage (figure 1).

Figure 1: A representative image of rat femur bone of the control group shows normal architecture represented by articular cartilage, trabecular bone, epiphyseal plate cartilage, and metaphyseal bone. Barcharts represents the measured thickness of articular cartilage, metaphyseal plate, and trabecular bone; data expressed as mean ± SD; *^#p<0.05 as compared to another group. C=Control group, Y=Yasmin group, M=Microgynon group, Mv=Marvelon group, D=Depo-Provera group.

Yasmin group has shown normal architecture of cortex of the cerebrum represented by neurons, glial cells, and mild vasogenic edema. Microgynon group has shown normal architecture of neurons, pericellular vacuoles of glial cells with vasogenic edema, and congested blood vessels. Marvelon group has shown normal architecture of neurons with gliosis, vasogenic edema, and congested blood vessels. Depo-Provera group has shown pericellular vacuoles, vasogenic edema, and congested blood vessels (figure2).

Figure 2: A representative image of the rat brain of the control group shows the normal architecture of the cortex of the cerebrum represented by neurons, glial cells, and blood vessels.

Ovary of Yasmin group shows normal architectures represented by primordial, primary, secondary, atretic, mature follicles, and corpus luteum with congested bold vessels. Ovary of Microgynon group shows degeneration of the cells of primary follicle, necrosis of the cells of secondary follicle, with the presence of atretic, mature follicles and corpus luteum with congested bold vessels. Ovary of Marvelon group shows necrosis of the cells of secondary follicle, with the presence of atretic follicle and corpus luteum with congested bold vessels. Ovary of Depo-Provera group shows the mass of increased fibrous connective tissue (Figure 3).

Figure 3: A representative image of the ovary of the control group shows normal architectures represented by primary, secondary, mature follicles, and corpus luteum. Barcharts represented Thickness of endometrium (X) and Follicle size, number, and maturation. Data expressed as mean±SD, *^p<0.05.

Cervix Yasmin group shows normal architectures represented by mucosa with stratified squamous epithelium, submucosa, and muscular layer. The Cervix of the Microgynon group shows normal architectures represented by mucosa with stratified squamous epithelium, muscularis layer, and submucosa infiltration of inflammatory cells. the cervix of the Marvelon group shows thickening of mucosa with stratified squamous epithelium, degeneration of epithelial cells of the mucosa. The Cervix of the Depo-Provera group shows severe necrosis and degeneration of stratified squamous epithelium of mucosa with the presence of necrotic debris in the lumen (figure 4).

Figure 4: A representative image of the rat cervix of the control group shows normal architectures represented by mucosa with simple columnar epithelium, submucosa, and muscularis layer. Score 0=Absent formation of inflammation, Score 1= Scanty amount of inflammation.

The mammary gland of the Yasmin group shows normal architectures of increased numbers and density of acini and ducts. Microgynon group shows necrosis and desquamation of epithelium lining ducts, hyperplasia of others and increased fibrous tissue surrounding ducts, infiltration of inflammatory cells in the lumen, and increased fibrous tissue surrounding. Marvelon group shows severe necrosis of epithelial cells lining ducts and desquamation of it in the lumen of ducts. Depo-Provera group shows severe necrosis of epithelial cells lining ducts and desquamation of it in the lumen of ducts (figure 5).

Figure 5: A representative image of the rat mammary gland of the control group shows normal architectures represented by acini and ducts.

Mammary gland, Yasmin has shown normal architectures of increased numbers and density of acini and ducts. Microgynon group has shown necrosis and desquamation of epithelium lining ducts, hyperplasia of others and increased fibrous tissue surrounding ducts, infiltration of inflammatory cells in the lumen, and increased fibrous tissue surrounding. Marvelon group has shown severe necrosis of epithelial cells lining ducts and desquamation of it in the lumen of ducts. Depo-Provera group has shown severe necrosis of epithelial cells lining ducts and desquamation of it in the lumen of ducts (figure 6).

Figure 6: A representative image of the rat mammary gland of the control group shows normal architectures represented by acini and ducts.

DISCUSSION:

In the present study, the histology of the endometrium, ovary, cervix, uterus, mammary glands, and bone were studied in rats after administration of oral contraceptives; Yasmin, Marvelon, Microgynon, and Depo-Provera injection. The study showed that Yasmin combined oral contraceptive does not affect the thickness of the endometrium as compared with control, while Depo-Provera injection caused a reduction of the thickness of endometrium as compared to that of the control group. All of them have induced a deleterious impact on bone architecture.

In a study done by Bhowmik and Mukher 1988, the administration of Depo-Provera reflected that the endometrium tissue of the rats' uterus steadily became quiescent and with continued hormonal therapy at a high dose, atrophy of the endometrium was reported⁹. Similarly, Bari and Choudhury 1977, reported that Depo-Provera injection reduced endometrium thickness as compared to the control group¹⁰. Atrophic change in the endometrium of the rats caused by Depo-Provera may be due to permanent progesterone stimulation from the drug Medroxyprogesterone to the endometrium in the absence of sufficient ovarian estrogen formation to afford ample endometrial priming¹⁰. Changes in the uterus of the rats observed in the present study with Depo-Provera were in agreement with studies and suggest that the drugs are capable of causing necrosis and atrophic changes in the endometrium¹¹.

Another study presented that users of Depo-Provera can exhibit several endometrial histological changes and the tissue finding do not correlate easily with the patient symptoms or duration of the medication use. Chronic endometriosis was a frequent finding in this study¹². In the present study, the Yasmin group showed an increase in the thickness of articular cartilage, metaphyseal plate, and trabecular bone while Depo-Provera showed a reduction in the thickness of articular cartilage, metaphyseal plate, and trabecular bone.

A study done by Walsh et al. 2008, reported that Depo-Provera use is conjoined with bone density defection at the spine and hip who used before bone peak mass, and concluded that Depo-Provera acts on the skeleton principally via estrogen defection¹³. A newer study conducted by Johnson et al. 2008, reported that Depo-Provera causes bone loss in women who use it for long period, but the loss appears largely reversible¹⁴.

Immunity status¹⁵, presence of breast cancer¹⁶, the status of renal function¹⁷, regular exercise and involvement in sport club¹⁸, association with polycystic ovary syndrome^19,20, presence of cardiac diseases²¹, and surgery into female genital tract²² have been reported as important parameters in women's overall health which might be associated with deleterious positive and negative effects on serum sex hormone levels and ultimately might lead to needs for hormonal replacement therapy²³

CONCLUSION:

The use of animal models is often an essential step to provide clear and wide knowledge about the adverse effect of an important widely used drug in our society. The current study showed that injectable type of contraceptives results in marked histological changes in the uterus and bone structure with lower to normal histological change with Yasmin oral contraceptive.

ACKNOWLEDGMENTS:

The authors are grateful to the College of Medicine and College of Veterinary Medicine in the University of Mosul for their provided facilities to conduct this research.

FUNDING:

Self-Funded.

ADHERENCE TO ETHICAL STANDARDS:

The study was approved by the Medical Research Ethics Committee at the University of Mosul.

CONFLICT OF INTEREST:

The authors declare that no conflict of interest exists for this research.

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Received on 18.03.2022 Modified on 21.04.2022

Research J. Pharm. and Tech 2023; 16(2):686-690.

DOI: 10.52711/0974-360X.2023.00117