Ovarian Cysts

Experimental animals
Prepubertal female Wistar rats (n=36) aged 4-6 weeks weighing 180-230 g were included in the experiment. In the Central Animal House (Seth Gordhandas Sunderdas (GS) Medical College and King Edward Memorial (KEM) Hospital, Mumbai, India), where controlled conditions were preserved with a temperature of 23°C±4°C and a humidity of 30%-70%, they were kept in standard laboratory conditions. Individual polypropylene cages (one per animal) with stainless steel top grills and amenities for delivering water and food were employed to keep the animals. In the cages, paddy husk served as bedding. Animals were provided with free access to UV-filtered water and pellet-based food (Chakan Oil Mills, Maharashtra, India). Twelve hourly light and dark cycles were maintained. The whole investigation was conducted under the standards established by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. Before the research began, approval from the Institutional Animal Ethics Committee (AEC/04/2017) was acquired.
Study drugs and chemicals
The test drugs, the ethanolic extracts of Caesalpinia crista (C. crista), were used as study drugs. They were obtained as gift samples from Alarsin Pharmaceuticals (Mumbai, India) with a certificate of analysis. Caesalpinia crista was a brown-colored soft ethanolic extract with an extractive value of 11% w/w. In Ayurveda texts, doses are mentioned in terms of the crude powder of Caesalpinia crista. The doses for the ethanolic extract were selected from the published literature [16 ,17 ] on C. crista and after consultation with experts in Ayurvedic medications and research. Oral administration of 100, 300, and 500 mg/kg of ethanolic extract was selected.
The chemicals used in the study were letrozole (used as an inducing agent) at a dose of 1 mg/kg, which was obtained from Sun Pharma (Mumbai, India) as a gift sample, and clomiphene citrate, which was used as an active (positive) control to treat PCOS. The dose used was 1.8 mg/kg, and it was purchased from Sigma-Aldrich, India. The vehicles used for the drugs were 1% carboxymethylcellulose (CMC) as the suspending agent for letrozole, and normal saline was used as the vehicle for the active control of clomiphene citrate and the test drugs (C. crista).
Experimental phase
Six groups in the experimental phase comprised six female rats each (Table 1). The female Wistar rats were randomly allocated to six groups with random code generated in Microsoft Excel (Microsoft Corp., Redmond, WA, USA). The rats were given inducing agents for 21 days (day 1-21), drugs were given for 15 days (day 22-36), and finally, blood and histopathology tissue samples were taken on day 37.
The vehicle control group was orally given 2 mL of 1% carboxymethylcellulose (CMC) for 21 days. CMC was used as the suspending agent for the inducing agent letrozole. This was followed by 15 days of 2 mL of normal saline, which was the vehicle for the study drugs and active control. The disease control group was given letrozole in the dose of 1 mg/kg orally, suspended in 2 mL of 1% CMC for 21 days. Following induction with letrozole, the rats were given 2 mL of normal saline for 15 days. The natural course of the disease, PCOS, was seen in this group.
The positive control group (clomiphene group) was given letrozole in the dose of 1 mg/kg orally, suspended in 2 mL of 1% CMC for 21 days. After this, the rats were given clomiphene citrate in a dosage of 1.8 mg/kg orally dissolved in 2 mL of normal saline for 15 days, and this group served as a comparator for the treatment with the study drugs. Low, medium, and high dosages of the study drug were administered to the treatment groups. These groups will be given letrozole in the dose of 1 mg/kg orally, suspended in 2 mL of 1% CMC for 21 days as an inducing agent for PCOS. This was followed by the study drug for 15 days in 2 mL of normal saline. The Caesalpinia crista ethanolic extract was administered orally for 15 days at dosages of 100 mg/kg, 300 mg/kg, and 500 mg/kg in 2 mL of normal saline to the low-, medium-, and high-dose groups.
Variables assessed
Vaginal smears were taken daily to provide information on estrous cyclicity at various intervals during the study. The smears were taken by the swab smear technique at 9 am every day. Once each week, a mono-pan digital weighing scale was used to record the rats’ total body weight. The weight was recorded every week in the morning. Blood glucose levels were recorded every week. Blood (1 mL) was collected every week from the rats by the retro-orbital method. To stop glycolysis, the blood was drawn into a bulb carrying sodium fluoride (10 mg/mL blood). These blood samples were used to determine the blood glucose levels by Trinder’s method. Ovulation was checked by counting the number of oocytes from each oviduct. Serum LH, FSH, and testosterone (T) estimations were done using the enzyme-linked immunosorbent assay (ELISA) method on day 37. The ELISA kits were procured from KinesisDx, USA. Histopathology of the ovaries was done to check for the number and size of the ovarian cysts. The dissected ovaries stored in 10% buffered formalin were processed to prepare paraffin blocks, and the slides were prepared. They underwent eosin and hematoxylin staining. The cystic and atretic follicle number was counted from each specimen. We also counted and compared the numbers of corpus luteum and the primary and secondary follicles between the groups.
Statistical analysis
Findings were presented as mean ± standard deviation (SD). A p-value of <0.05 was considered statistically significant. The blood glucose within the group for the two groups was compared using repeated measures analysis of variance (ANOVA) during standardization. The variables body weight, blood glucose, vaginal smears, ovulation, serum testosterone, serum LH, and serum FSH were compared between the groups using one-way ANOVA with post hoc Tukey-Kramer multiple comparisons test. p<0.05 was considered significant. The statistical analysis software GraphPad InStat (Graphpad Software Inc., San Diego, CA, USA) version 3.06 was utilized.