ABSTRACT
Purpose
Procarbazine, an alkylating agent, depletes ovarian reserve. Omegaven exhibits anti‐apoptotic, anti‐inflammatory, and antioxidant properties. We aimed to investigate the preventative benefits of Omegaven against the deleterious effects of Procarbazine on the ovaries.
Methods
Thirty female rats were categorized into groups: Control, n = 6; Omegaven, n = 8; Procarbazine, n = 8; and Procarbazine+Omegaven, n = 8. Biochemical and histological analyses were conducted.
Results
Total follicle count calculated from evaluated sections is significantly reduced in the Procarbazine group compared to other groups. Immunoreactivities of inflammatory and apoptotic markers were significantly elevated in the Procarbazine group and were diminished significantly in the Procarbazine+Omegaven group. In the Procarbazine group, ultrastructural alterations due to atresia were marked by the presence of lipid droplets, multivesicular bodies, and eosinophil infiltration. The ultrastructural alterations were less pronounced in the Procarbazine + Omegaven group. In the Procarbazine group, prooxidative, inflammatory, and apoptotic markers in ovarian tissue were elevated, while antioxidative indicators were significantly diminished. Blood chemistry analyses yielded comparable results. AMH levels were markedly reduced in the Procarbazine Group compared to the control group. The AMH levels in the Procarbazine + Omegaven group exhibit a rise significantly in blood serum samples.
Conclusions
Omegaven is a promising pharmacological agent for mitigating the deleterious effects of Procarbazine on ovarian reserve.
Keywords: alkylating agents, chemotherapy, omega‐3, ovarian reserve, procarbazine
1. Introduction
Women previously exposed to chemotherapeutics may experience premature ovarian insufficiency, diminished ovarian reserve, early menopause, and infertility [1]. Literature on sustaining fertility post‐chemotherapy has not progressed beyond advising early delivery [2, 3]. Techniques like cryopreservation, involving the extraction and freezing of ovarian tissue prior to treatment, followed by thawing and transplantation back to the patient, have been addressed in scholarly articles but are still experimental and not broadly implemented [4].
Alkylating agents are among the chemotherapeutic medication categories that most adversely affect ovarian reserve [5]. Alkylating compounds are recognized for their detrimental impact on ovarian reserve via chronic inflammation, apoptosis, and oxidative pathways [6, 7, 8]. Procarbazine, an alkylating chemotherapeutic agent, has demonstrated a dose‐dependent detrimental effect on ovarian reserve [9, 10].
In Procarbazine‐treated rats, Quercetin [11] decreased plasma oxidative markers and showed beneficial effects on liver tissues. Resveratrol has been utilized in cell culture research to mitigate the harmful effects of Procarbazine on bone marrow cells by reducing DNA damage [12]. Melatonin [13] and Clomiphene Citrate [14] have been utilized experimentally to mitigate the damage inflicted by Procarbazine on testicular tissue, which has a physiology analogous to that of ovarian tissue.
Omegaven is an omega‐3 fatty acid administered parenterally in humans. It is utilized safely in whole parenteral feeding. It has been utilized safely in cancer patients and has demonstrated an enhancement in immune response [15].
Omegaven has demonstrated the ability to diminish apoptosis in both the intestinal ischemia–reperfusion model [16] and the pulmonary ischemia–reperfusion paradigm [17]. In the intestinal ischemia–reperfusion model [16], it was observed that the intestinal tissue of rats administered Omegaven exhibited preservation in volume and microscopic morphology, along with a reduction in apoptosis. In the acute pulmonary ischemia–reperfusion model [17], Omegaven was observed to diminish inflammation and apoptosis while positively influencing liver functioning.
A retrospective investigation revealed that Omegaven infusion in patients with Ulcerative Colitis, Crohn's disease, or chronic intestinal insufficiency resulted in statistically significant healing in inflammatory markers [18].
In an experimental research, Omegaven enhanced oxidative parameters in endotoxic shock and showed protective effects on cardiac and intestinal tissues [19].
This study sought to ascertain the potential protective effects of Omegaven, an economical and safe drug used in clinical settings for cancer patients, against the deleterious effects of Procarbazine, an alkylating agent, on the ovary.
2. Materials and Method
The current research received approval from the Local Ethics Committee for Animal Experiments at Cukurova University (Meeting No: 7, Date: 07/10/2022, Decision No: 2).
2.1. Statistical Power Assessment
No research exists in the literature that seeks to ascertain the potential preventive effects of Omegaven against the deleterious effects of Procarbazine on the ovaries. Consequently, it was determined to incorporate a total of 24 rats, with 6 assigned to each group, based on the high effect size (d = 0.80), 80% power, and 5% margin of error recommended by Cohen using G*Power 3.1.9.7. Given the anticipated 25% mortality rate among rats in the procarbazine, procarbazine+omegaven, and omegaven groups, the study was executed with a total of 30 rats, allocating 8 rats to each of these groups.
2.2. Animal Derivative
This study utilized 30 young adult female Wistar Albino rats, aged 4–6 months, with an average weight of 200–250 g. Throughout the trial, the animals were accommodated at the ÇÜ‐SABİDAM Unit, with no restrictions on water or feed provided. All animals in the study were maintained under a 12‐h light and 12‐h dark cycle. The rats in the study were systematically categorized into groups based on their age and weight. To synchronize the estrus cycles of the groups, one rat from each group was cohabitated in the same cage for 1 week. On the eve of the trial, an assessment was conducted to determine if the rats exhibited estrus synchrony. Vaginal swabs were collected from the rats using a moist cotton swab, which was subsequently examined microscopically [20]. The groups were allocated in a balanced manner based on their estrus stages.
2.3. Pharmaceuticals and Dosages
Procarbazine: The trial utilized Natulan in 50 mg tablet form. The capsule's contents were dissolved in distilled water and readied for gavage administration. The dosage of Procarbazine administered in several rat tests was 2 mg/kg/day for either 1 week or 2 weeks, or 62.5 mg/kg once weekly for 4 weeks. The approach most analogous to human application was used in the study [11], and it was intended to administer 2 mg/kg/day to rats for 1 week.
Omegaven: The dosage was determined based on trials in which chronic daily administration of omegaven was conducted at a rate of 1 mL/kg/day via intraperitoneal injection [21].
2.4. Groups
Control Group, n = 6: Rats in this cohort received 2 mL of distilled water via gavage every day during the initial week and 1 mL/kg of intraperitoneal saline every day throughout the first and second week.
The Omegaven Group, n = 8: Rats in this cohort were administered 2 mL of distilled water daily via gavage for the initial week, and 1 mL/kg of Omegaven intraperitoneally each day for the first and second weeks.
Procarbazine Group, n = 8: Rats in this cohort received 2 mg/kg/day of Procarbazine for the initial week via gavage and 1 mL/kg of intraperitoneal saline every day for the first and second weeks.
Procarbazine + Omegaven Group, n = 8: Rats in this cohort were administered 2 mg/kg/day of Procarbazine daily via gavage throughout the initial week and 1 mL/kg of Omegaven intraperitoneally on a daily basis for the first and second weeks.
2.5. Anesthesia, Surgical Procedures, Euthanasia
An intraperitoneal administration of a combination comprising 85 mg/kg ketamine hydrochloride and 15 mg/kg xylazine was performed for abdominal surgery at a dosage of 0.5–1 mL/kg.
All rats were weighed prior to anesthesia and administered an appropriate anesthetic dose. Once anesthesia was established, they were positioned dorsally, and a 2 cm midline incision was made in the abdomen. The ovarian tissues were clamped, blood was extracted from the abdominal aorta, euthanasia was conducted, and both ovaries were excised. The first clamping of the ovarian tissues was performed to ensure that the subsequent events during blood collection would not impact the ovarian tissue. The rationale for collecting blood without excising the ovarian tissue was to mitigate the risk of insufficient blood collection resulting from hemorrhage during the tissue removal process.
The right ovaries of the rats were dispatched to the histology laboratory, while the left ovaries were forwarded to the biochemistry laboratory.
2.6. Histopathological Examination
2.6.1. Vaginal Cytology
The phases of the estrous cycle in rats were established before the experiment. Vaginal smears were stained with carbol fuchsin and analyzed using an Olympus BX 53 light microscope (Tokyo, Japan) [20]. The rats were in the estrous phase, marked by predominantly anucleated cornified cells at the experiment's onset (Figure 1).
FIGURE 1.
(A–D) The estrous cycle phase of the rats before the experiment. Rats were in the estrous phase characterized by primarily anucleated cornified cells. Carbol fuchsin stain. Bars = 50 μm.
2.6.2. Light Microscopy
The ovaries were fixed in 10% neutral formalin for 72 h for light microscopy analysis. Subsequently, standard tissue preparation was conducted utilizing the Leica TP1020 (Germany) automatic tissue processor. Sections of 5 μm thickness were taken from the paraffin blocks and subsequently stained with Hematoxylin and Eosin (H&E). Sections of each animal were taken at least 80 μm apart to prevent counting the same follicle again [22]. Four sections were obtained for each animal to count follicles. All follicles in every section were counted and compared with each other for ovarian reserve estimation. One experimenter performed the procedure blind to the treatment group. Statistical analyses were performed between the counted sections of the groups. Follicles were categorized as primordial follicles, characterized by an oocyte encircled by squamous follicle cells. Primary follicles contained an oocyte encircled by one or more layers of granulosa cells. Secondary follicles had a layer of granulosa cells with the development of small fluid‐filled cavities inside them. Mature Graafian follicles included a substantial antrum and an oocyte within the cumulus oophorus. The corpora lutea comprised lutein cells. Atretic follicles exhibited abnormal features, including degenerated oocytes, infiltration of the granulosa layer by neutrophils and macrophages, vascularized connective tissue, separation of the granulosa cell layer from the zona pellucida, as well as degeneration and apoptosis within the granulosa cell layer. Total follicle count was calculated by adding primordial, primary, secondary, tertiary, and Graafian follicles. The slices were analyzed and photographed using an Olympus BX 53 light microscope (Tokyo, Japan).
2.6.3. Immunohistochemical Techniques
For immunohistochemical examination, 5 μm‐thick slices were deparaffinized, immersed in a citrate solution (pH: 6) in a 95°C water bath for 30 min, and subsequently incubated with 3% H2O2 to inhibit endogenous peroxidase for 15 min at room temperature. Tissue slices were incubated with blocking reagent (IHC Kit, ab93705, Abcam, USA) for 15 min. Primary antibodies, specifically TNF alpha antibody (AF7014, Affinity Biosciences), IL6 antibody (DF6087, Affinity Biosciences), and Caspase 3 antibody (E‐AB‐13815, Elabscience), were added and incubated overnight at 4°C. Subsequent to washing the specimens the following day, the secondary antibody (IHC Kit, ab93705, Abcam, MA, USA) was administered, followed by a 10‐min staining with 3‐amino‐9‐ethylcarbazole (AEC IHC Kit ab93705, Abcam, MA, USA) and a 30‐s application of hematoxylin reagent prior to dehydration. Stained section images were examined using light microscopy (Olympus BX53, Tokyo, Japan).
For immunohistochemical analyses, the H scoring system was used. The immunoreactivities of TNFα, IL‐6, and caspase 3 were evaluated in 3 representative sections at least 80 μm apart per animal, and median values were calculated per animal for 6 animals in all groups at ×40 magnification. One experimenter performed the procedure blind to the treatment group. The H score was calculated by summing the product of the four different staining intensities with the percentage of positive cells as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The formula ‘I (intensity) × PC (percentage of positive cells)’ was used to calculate the H score value. Statistical analyses were performed among the animals of the groups.
2.6.4. Electron Microscopy
The ovarian specimens for electron microscopy were fixed for 4 h in 5% glutaraldehyde in Millonig phosphate buffer at pH 7.4, followed by post‐fixation with 1% osmium tetroxide in the same buffer for 2 h at 4°C. Samples were dehydrated using a graded series of ethanol and subsequently embedded in araldite. Semi‐thin sections were prepared using a Reichert Ultracut S ultramicrotome, stained with toluidine blue, and suitable areas for electron microscopic examination were identified. Thin sections were taken from the designated locations and stained with uranyl acetate and lead citrate. Subsequently, they were examined and imaged using a Jeol JEM 1400 transmission electron microscope.
2.7. Biochemical Analysis
The biochemical examination involved preparing tissue homogenates by sectioning them into minute fragments and washing them in ice‐cold PBS (0.01 M, pH 7.4) to remove excess blood. A tissue weight to PBS volume ratio of 1:9 was used to homogenize the samples in PBS on ice after weighing. The homogenates were centrifuged at +4°C at 5000 × g for 10 min to isolate the supernatant, which was stored at −86°C until analysis.
For serum specimen preparation, rat blood was coagulated for 20 min at ambient temperature, centrifuged at 3000 RPM for 20 min, and stored in Eppendorf tubes at −86°C until analysis.
2.8. Measurement of AMH Levels
AMH concentrations were measured using the Elabscience Sandwich‐ELISA kit (Catalog No. E‐EL‐R3022). The methodology involved adding samples and standards to the plates, incubating them, and then applying biotinylated antibody and HRP conjugate and washing. After adding substrate and stop solutions, absorbance was measured at 450 nm and calculated.
2.9. Measurement of Estradiol Levels
Estradiol concentrations were evaluated by competitive ELISA, featuring sample E2 competing with immobilized antigen for antibody binding, followed by HRP‐TMB detection and spectrophotometric analysis at 450 nm.
2.9.1. Measurement of Apoptotic and Inflammatory Markers (TNFα, IL‐6, Caspase‐3)
The concentrations were compared to a calibration curve. Using BT LAB ELISA Kits (Cat. No E0764Ra, E0135Ra, E1648Ra), apoptotic and inflammatory biomarkers (TNFα, IL‐6, Caspase‐3) were quantified. Each reagent, sample, and standard was made according to the kit protocol. After adding sample and ELISA reagent to each well, they incubated at 37°C for an hour. Five wash buffer rinses followed. Add 50 μL of substrate solutions A and B and incubate at 37°C for 10 min. After incubation, a stop solution was added to check color development. Calculations were made by measuring the optical density at 450 nm in 10 min.
2.10. Oxidative Parameter Assessment
Ovarian tissue homogenate and blood were tested for TAS, TOS, SOD, GSH‐Px, and MDA. The average was calculated from triplicate samples. Principles of serum and homogenate oxidative parameter detection:
TAS: A sufficient oxidant oxidizes ABTS to green ABTS•+, which antioxidants decrease to colorless ABTS. ABTS+ absorbance at 660 nm indicates the sample's total antioxidant status (TAS). Trolox is a vitamin E mimic with similar antioxidant properties. A complete antioxidant status reference chemical is Trolox.
The oxidizing agent in the sample converts Fe2+ to Fe3+ under acidic circumstances, forming a strong combination with xylenol orange, resulting in a blue‐purple color. At pH 2–3, the maximum absorption wavelength is 590 nm, and the color intensity correlates with the concentration of oxidizing agents over a defined duration, allowing indirect computation of the sample's overall oxidation state. Oxidative Stress Index (OSI) is calculated by the formulation: OSI: (TOS/TAS)*100 [23].
This kit measured SOD activity using the WST‐1 method, based on the following principles. By reacting WST‐1 with O2 •‐, Xanthine Oxidase (XO) creates a water‐soluble formazan dye. SOD disproportionates superoxide anions, which may slow the reaction. SOD activity is inversely related to formazan dye.
GSH‐Px helps convert hydrogen peroxide (H2O2) and reduced glutathione into water (H2O) and oxidized glutathione (GSSG). Glutathione peroxidase activity is measured by reaction rate. Glutathione activity is measured by reduced glutathione depletion. Hydrogen peroxide (H2O2) and reduced glutathione interact without GSH‐Px catalysis; hence non‐enzymatic GSH reduction must be neglected. GSH reacts with dinitrobenzoic acid to form the yellow 5‐thio‐dinitrobenzoic acid anion. Measure 412 nm absorbance to calculate GSH concentration.
Competitive ELISA is used in the MDA ELISA kit. This package comprises an MDA‐precoated micro ELISA plate. MDA in samples or standards competes with a specified amount of MDA on the solid‐phase support for binding sites on the MDA‐specific Biotinylated Detection Antibody. The plate is cleaned of excess conjugate and unbound samples or standards, then Avidin conjugated to HPR is applied to each microplate well and incubated. TMB substrate solution is then added to each well. The enzyme‐substrate interaction is ended with a stop solution, and the color change is measured at 450 ± 2 nm using spectrophotometry. Sample MDA concentration is calculated by comparing optical density to the standard curve.
2.11. Statistical Examination
All analyses were conducted utilizing IBM SPSS Statistics Version 20.0 software (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.). The Shapiro–Wilk test was employed to evaluate the normality of continuous variable distributions. Continuous variables were summarized as mean and standard deviation and as median and minimum–maximum where appropriate. ANOVA was employed to examine more than two groups of normally distributed data, while the Bonferroni test was utilized for multiple group comparisons. The Kruskal–Wallis test was employed to compare more than two groups of non‐normally distributed data, whereas the Dunn test was utilized for multiple group comparisons. The statistical significance threshold for all tests was set at 0.05.
3. Results
3.1. Histopathological Findings
3.1.1. Results From Light Microscopy
The ovaries of the control group were covered with simple cuboidal surface epithelium and comprised of cortex and medulla. Primordial follicles, follicles at various developmental stages, and corpora lutea were observed to be morphologically normal in the cortex. The Procarbazine group exhibited a substantial reduction in primordial follicle counts compared to the Control and Omegaven groups. The number of primary follicles was significantly reduced in the Procarbazine group compared to the Control group. No substantial difference was observed in the atretic follicle counts among the groups. Enhanced congestion in the stroma was seen in the Procarbazine group compared to the other groups. The Procarbazine + Omegaven group had a substantial increase in primordial follicle counts compared to the Procarbazine group. The total follicle count in evaluated sections was markedly reduced in the Procarbazine group relative to the Control group, whereas it dramatically increased in the Procarbazine + Omegaven group compared to the Procarbazine group (Figure 2 and Table 1).
FIGURE 2.
Light microscopic examination of the experimental groups (H&E). (A) Control group is seen in normal morphology. (B) Procarbazine group. (C) Procarbazine + Omegaven group. (D) Omegaven group. Surface epithelium (SE), primordial follicle (Prf), primary follicle (Pf), secondary follicle (Sf), atretic follicle (Atf), corpus luteum (CL) and congession (*) are indicated. Bars = 100 μm.
TABLE 1.
Follicle numbers in the ovarian tissue sections.
| Control | Omegaven | Procarbazine | Procarbazine + Omegaven | p | |
|---|---|---|---|---|---|
| Primordial | 9.8 ± 1.3 a | 11.3 ± 2.1 a , b | 5.8 ± 1.1 b | 9.1 ± 1.9 | < 0.001 |
| Primary | 5.5 ± 1.2 | 5.0 ± 0.9 | 4.3 ± 1.4 b | 6.3 ± 1.5 | 0.006 |
| Secondary | 1.0 (0.0–4.0) | 2.0 (0.0–3.0) | 1.0 (0.0–3.0) | 1.0 (0.0–4.0) | 0.295 |
| Tertiary | 0.0 (0.0–2.0) | 1.0 (0.0–2.0) | 0.0 (0.0–1.0) | 0.0 (0.0–1.0) | 0.152 |
| Corpus Luteum | 6.0 (4.0–9.0) b | 6.5 (3.0–9.0) | 5.0 (3.0–11.0) b | 8.0 (6.0–11.0) | 0.006 |
| Atretic follicles | 6.0 (4.0–8.0) | 5.0 (4.0–6.0) | 5.0 (3.0–8.0) | 6.0 (4.0–8.0) | 0.228 |
| Primary + secondary | 6.8 ± 2.0 | 6.8 ± 1.4 | 5.3 ± 1.4 b | 7.7 ± 2.4 | 0.030 |
| Total follicle count | 23.3 ± 3.4 a | 25.5 ± 2.9 a | 17.2 ± 2.6 b | 25.5 ± 3.6 | < 0.001 |
Note: Data were expressed as mean ± standard deviation or median (min–max).
p < 0.05 comparison between Procarbazine.
p < 0.05 comparison between Procarbazine + Omagaven.
3.1.2. Immunohistochemical Findings
Immunoreactivities of TNFα and IL‐6 were significantly increased in the Procarbazine group compared to the Control and Omegaven groups and were significantly reduced in the Procarbazine + Omegaven group compared to the Procarbazine group. The Omegaven group had markedly reduced immunoreactivities of TNFα and IL‐6 in comparison to the Control group. Caspase‐3 immunoreactivity was elevated in the Procarbazine group relative to the Omegaven group and considerably diminished in the Procarbazine + Omegaven group compared to the Procarbazine group. The immunoreactivities of Caspase 3 in the Omegaven group were significantly lower than those in the Control group (Figure 3 and Table 2).
FIGURE 3.
TNF alpha, IL6 and caspase 3 immunoreactivities of the experimental groups. Bars = 100 μm.
TABLE 2.
Immunohistochemical stainings of the groups.
| Control | Omegaven | Procarbazine | Procarbazine+ Omegaven | p | |
|---|---|---|---|---|---|
| Caspase 3 | 0.41 (0.39–0.42) a | 0.28 (0.28–0.33) b | 0.61 (0.58–0.64) c | 0.38 (0.35–0.40) | < 0.001 |
| TNF alpha | 0.47 ± 0.02 a , b | 0.34 ± 0.02 b , c | 0.86 ± 0.02 c | 0.45 ± 0.02 | < 0.001 |
| IL6 | 0.39 ± 0.02 a , b | 0.32 ± 0.02 b , c | 0.59 ± 0.02 c | 0.41 ± 0.02 | < 0.001 |
Note: Data were expressed as mean ± standard deviation or median (min–max).
p < 0.05 comparison between Omagaven.
p < 0.05 comparison between Procarbazine.
p < 0.05 comparison between Procarbazine + Omagaven.
3.1.3. Results From Electron Microscopy
The Control group had normal ovarian surface epithelium, primordial follicles, developing follicles, corpora lutea, stroma, and some atretic follicles (Figure 4).
FIGURE 4.
Electron micrographs of control group. (A) Surface epithelium is seen normally. Bar = 0.1 μm. (B) Primordial follicle on the left upper corner, stroma and a growing follicle on the right side are seen normally. Bar = 1 μm. (C) Primordial follicle is observed normally. Bar = 2 μm. (D) Secondary follicle is seen normally. Bar = 0.2 μm. (E) Granulosa cells of an atretic follicle is seen. Bar = 0.2 μm. (F) Corpus lutheum is observed normally. Bar = 0.2 μm. Nucleus (N), oocyte (O), follicle cells (FC), granulosa cells (GC) and zona pellucida (ZP) are indicated.
The Procarbazine group exhibited enlargement of granular endoplasmic reticulum cisternae and perinuclear cisternae, vacuoles, an increase in lysosomes and lipid droplets in the ovarian surface epithelium, as well as increased collagen fibers in the tunica albuginea (Figure 5A,B). Increased lysosomal presence and swollen mitochondria in the oocytes of primordial follicles (Figure 5C) were observed, together with atresia marked by multivesicular structures in the primordial follicles throughout several fields (Figure 5D). Developing follicles exhibited increased lipid droplets, lysosomes, and vacuoles within the oocyte, along with an expanded perivitelline space. The basement membrane separating the granulosa cells from the theca interna exhibited increased thickness, resulting in a wavy layer (Figure 5E–G). Swollen mitochondria and enlarged smooth endoplasmic reticulum cisternae were observed in the luteal cells of the corpus luteum (Figure 5H). Multivesicular structures were identified in the stromal cells (Figure 5I), along with eosinophils and apoptotic cells in the stroma (Figure 5J,K).
FIGURE 5.
Electron micrographs of Procarbazine group. (A and B) Surface epithelium (SE). Enlargement of granular endoplasmic reticulum cisternae (GER) and perinuclear cisternae (*), vacuoles (V), increased lysosomes (Ly) and lipid droplets (L) in the ovarian surface epithelium, increased collagen fibers (Coll) in the tunica albuginea (TA) are seen. Bars = 0.2 μm. (C) Increased lysosomes (Ly) and swollen mitochondriae (M) in the oocyte (O) of a primordial follicle is seen. Follicle cells (FC). Bar = 0.1 μm. (D) An atretic primordial follicle filled with multivesicular bodies (Mvb) is seen. Bar = 0.2 μm. (E) Vacuoles (V) and lysosomes (Ly) in a growing follicle oocyte (O) are observed. Bar = 0.2 μm. (F) Atretic growing follicle reveals increased lipid droplets (L), lysosomes (Ly) and vacuoles (V) in the oocyte and increased perivitelline space (Pvs). Bar = 1 μm. (G) Increased vacuoles (V) in the oocyte and increased perivitelline space (Pvs) are seen. The basal lamina (Bl) is increased in thickness forming a wavy layer. Bar = 1 μm. (H) Lipid droplets (L), swollen mitochondriae (M) and enlargement of smooth endoplasmic reticulum cisternae (SER) in the corpus luteum are seen. Bar = 0.1 μm. (I) Stromal cells (S) reveal multivesicular bodies (Mvb). Bar = 2 μm. (J and K) Eosinophils (Eo) and apoptotic cells (Ap) are observed in the stroma. Bars = 0.2 μm.
The ultrastructural alterations were diminished in the Procarbazine + Omegaven group (Figure 6).
FIGURE 6.
Electron micrographs of Procarbazine + Omegaven group. (A) Surface epithelium (SE) and tunica albuginea (TA) are seen normally. Bar = 0.1 μm. (B) Swollen mitochondriae (M) and vacuoles are seen in a primordial follicle oocyte (O). Follicle cells (FC) are seen normally. Bar = 0.1 μm. (C and D) Primary follicles with their oocytes (O) and granulosa cells (GC) are observed normally. Stroma (S) is seen normally. Bars = 0.1 μm and 0.2 μm. (E) Granulosa cells (GC), basal lamina (Bl) and theca interna (TI) are observed normally in a secondary follicle. Bar = 1 μm. (F) Corpus luteum is observed normally. Nucleus (N). Bar = 0.1 μm.
The ovarian surface epithelium in the Omegaven group exhibited a normal structural morphology. Primordial follicles had lysosomes and vacuoles within the oocyte, with certain primordial follicles including multivesicular structures. Primary and secondary follicles had normal morphology. Vacuoles were observed in the cytoplasm of certain secondary follicle oocytes. Increased lysosomes and multivesicular bodies were seen in the stromal cells (Figure 7).
FIGURE 7.
Electron micrographs of Omegaven group. (A) Surface epithelium (SE) is seen normally. Bar = 1 μm. (B) Increased lysosomes (Ly) and vacuoles (V) are seen in the primordial follicle oocyte (O). Nucleus (N). Bar = 0.2 μm. (C) A primordial follicle filled with multivesicular bodies (Mvb) is seen. Bar = 2 μm. (D) Oocyte (O), zona pellucida (ZP) and granulosa cells (GC) are observed normally in a secondary follicle. Bar = 0.2 μm. (E) Granulosa cells (GC) and zona pellucida (ZP) are seen normally in a secondary follicle. Oocte (O) reveals vacuoles (V) in the cytoplasm. Nucleus (N). Bar = 2 μm. (F) Increased lysosomes (Ly) and multivesicular bodies (Mvb) are seen in a stromal cell. Bar = 0.2 μm.
3.2. Biochemical Findings
The biochemical findings of tissue homogenates (Table 3) and blood serums (Table 4) for the groups are summarized in the respective tables. The AMH levels in the Procarbazine group were markedly reduced in both tissue (Table 3) and serum (Table 4) compared to the control group. Omegaven treatment significantly inhibits the reduction of AMH levels in serum. It also partially restored tissue AMH levels, albeit not to a statistically significant extent. Although no statistically significant difference exists among groups for tissue estradiol levels, the serum estradiol level in the Procarbazine group is significantly elevated compared to the Procarbazine + Omegaven group. The current investigation fails to reveal noteworthy findings regarding oxidative and antioxidative pathways, with the exception of MDA and GSH‐Px. The MDA tissue level is markedly elevated, whereas the GSH‐Px level is considerably diminished in the Procarbazine group compared to all other groups. Rats subjected to Omegaven exhibited reduced tissue MDA levels in comparison to the control group. The results for serum MDA and GSH‐PX were consistent with these findings. Inflammatory and apoptotic parameters yield significant results among groups, aligning with our immunohistochemistry findings. Both inflammatory and apoptotic measures exhibit considerable degradation in the Procarbazine group, but Omegaven treatment dramatically ameliorated the outcomes.
TABLE 3.
Biochemical findings of tissue homogenates of groups.
| Control (n = 6) | Omegaven (n = 8) | Procarbazine (n = 8) | Procarbazine + Omegaven (n = 8) | p | |
|---|---|---|---|---|---|
| TOS | 29.5 (28.9–34.3) a , b , c | 37.4 (35.5–43.8) | 38.7 (33.5–43.6) | 37.8 (31.4–43.1) | 0.005 |
| TAS | 9.9 (9.6–9.9) | 8.9 (8.1–10.5) b | 10.5 (9.3–11.1) | 9.8 (8.1–10.8) | 0.039 |
| OSI | 301.0 (291.9–346.5) a , c | 417.8 (358.1–487.7) | 373.4 (324.1–420.4) | 397.4 (350.9–448.9) | 0.001 |
| T‐SOD | 464.8 (447.1–472.0) | 489.5 (468.0–518.4) b | 443.9 (415.4–483.8) | 483.1 (415.4–497.0) | 0.003 |
| MDA | 75.8 ± 3.2 a , b , c | 68.9 ± 3.5 b , c | 84.3 ± 2.9 c | 60.9 ± 2.4 | < 0.001 |
| GSHPx | 163.8 ± 8.7 b , c | 137.9 ± 21.1 b | 94.9 ± 23.1 c | 126.8 ± 9.9 | < 0.001 |
| TNFalpha | 23.8 ± 5.6 b | 41.1 ± 3.2 b | 269.5 ± 29.9 c | 45.5 ± 4.9 | < 0.001 |
| IL6 | 6.7 (6.1–7.8) | 6.3 (5.8–7.3) b | 8.9 (7.4–10.8) c | 6.1 (5.5–6.3) | < 0.001 |
| Caspase3 | 3.7 (2.6–9.6) | 2.4 (1.8–3.6) b | 15.3 (11.1–24.6) c | 2.2 (1.8–4.2) | < 0.001 |
| AMH (pg/mL) | 3162.0 (3029.0–3202.0) b , c | 2869.0 (2762.0–2989.0) | 2199.0 (1535.0–2995.0) | 2761.0 (2305.0–2997.0) | < 0.001 |
| E2 (pg/mL) | 42.7 ± 6.4 | 45.2 ± 6.7 | 53.1 ± 10.7 | 44.9 ± 9.8 | 0.137 |
Note: Data were expressed as mean ± standard deviation or median(min–max). Units used: TOS: μmol H2O2 Equiv. /L, TAS: μmol Trolox Equiv. /L, T‐SOD: U/mL, MDA: ng/mL, GSHPx: U/mL, IL6: ng/L, Caspase 3: ng/m AMH: pg/mL, E2: pg/mL.
p < 0.05 comparison between Omagaven.
p < 0.05 comparison between Procarbazine.
p < 0.05 comparison between Procarbazine + Omagaven.
TABLE 4.
Biochemical findings of blood serums of groups.
| Control (n = 6) | Omegaven (n = 8) | Procarbazine (n = 8) | Procarbazine + Omegaven (n = 8) | p | |
|---|---|---|---|---|---|
| TOS | 55.8 (52.0–72.1) c | 82.8 (49.7–94.7) | 56.3 (51.8–64.0) c | 81.2 (62.5–97.9) | 0.004 |
| TAS | 3.4 ± 0.7 | 3.2 ± 0.2 | 3.4 ± 0.4 | 3.2 ± 0.2 | 0.708 |
| OSI | 1646.3 (1544.1–2495.5) c | 2595.4 (1506.1–3156.7) | 1684.6 (1461.5–1882.4) c | 2497.6 (1953.1–3158.1) | 0.003 |
| T‐SOD | 92.9 (84.4–103.8) | 107.8 (100.8–109.4) b | 84.1 (74.0–90.7) c | 108.6 (102.2–113.0) | < 0.001 |
| MDA | 68.6 ± 9.3 a | 57.8 ± 4.8 b | 77.0 ± 2.7 c | 50.3 ± 8.2 | < 0.001 |
| GSHPx | 388.7 ± 152.8 b | 298.6 ± 38.4 | 218.0 ± 104.4 | 260.2 ± 92.0 | 0.029 |
| TNFalpha | 185.2 ± 38.3 b | 198.6 ± 28.0 b | 267.4 ± 18.4 c | 200.1 ± 17.1 | < 0.001 |
| IL6 | 14.6 ± 1.9 b | 16.3 ± 2.2 b | 59.7 ± 2.8 c | 14.1 ± 1.8 | < 0.001 |
| Caspase3 | 9.3 ± 0.3 b | 8.6 ± 0.8 b | 11.0 ± 0.8 c | 9.1 ± 1.3 | < 0.001 |
| AMH | 2394.8 ± 177.1 b | 2061.3 ± 369.9 | 1874.4 ± 204.9 | 2216.1 ± 344.7 | 0.018 |
| E2 | 34.9 ± 7.5 | 39.9 ± 4.5 | 41.2 ± 2.7 c | 36.2 ± 2.8 | 0.040 |
Note: Data were expressed as mean ± standard deviation or median(min–max). Units used: TOS: μmol H2O2 Equiv. /L, TAS: μmol Trolox Equiv. /L, T‐SOD: U/mL, MDA: ng/mL, GSHPx: U/mL, IL6: ng/L, Caspase 3: ng/m AMH: pg/mL, E2: pg/mL.
p < 0.05 comparison between Omagaven.
p < 0.05 comparison between Procarbazine.
p < 0.05 comparison between Procarbazine + Omagaven.
4. Discussion
The findings from the follicular count of ovarian sections and AMH levels in this study indicate that Procarbazine markedly diminishes ovarian reserve in rats exposed to Procarbazine, and Omegaven mitigates this effect significantly. The total follicle count in evaluated sections and serum AMH levels in the Procarbazine + Omegaven group are comparable to those in the control group. This is, to our knowledge, the first investigation demonstrating an agent that confers protective benefits on the ovarian reserves exposed to Procarbazine.
Inflammation markedly escalated in the Procarbazine group relative to all other groups, while administration of Omegaven to procarbazine‐exposed rats normalized inflammation levels, suggesting it may serve as an effective agent to safeguard ovaries against the deleterious effects of procarbazine. Chemotherapy has been demonstrated to diminish ovarian reserve due to persistent inflammation [8]. Our current study yielded analogous findings.
Apoptosis is markedly elevated in the Procarbazine group compared to the Procarbazine + Omegaven group. According to biochemical and immunohistochemical findings, the addition of Omegaven can be viewed as safeguarding ovarian tissue against apoptosis. Indeed, a primary distinction between apoptosis and atresia is the presence of inflammation. In the presence of inflammation, it is atresia rather than apoptosis. Our observations suggest that Procarbazine induces atresia rather than apoptosis. According to the literature [7], inhibiting apoptosis diminishes the deleterious effects of alkylating agents on tissue. We also identified elevated apoptotic markers in the Procarbazine group using biochemical and immunohistochemical investigation.
Several oxidation and antioxidation pathway results in blood serum and tissue were measured. While significant adverse changes were seen in lipid peroxidation pathways like MDA and GSH‐Px in tissue homogenates and serum in the Procarbazine group, parameters not directly related to lipid peroxidation like T‐SOD, TAS, TOS were not significantly affected. Omegaven treatment to Procarbazine‐exposed rats improved MDA levels significantly in serum and tissue and significantly improved GSH‐Px levels in tissue. GSH‐Px levels also increased in the serum of the Procarbazine + Omegaven group but did not reach significance. The ovaries are a fundamental organ in the body responsible for the production of lipid hormones. There may be a correlation between the pathophysiology of Procarbazine‐induced ovarian damage and hormonal metabolism. Alp et al. [13] investigated the testicles of male rats treated with Procarbazine and notably observed analogous alterations in MDA and GSH‐Px levels in their findings. Testicles, akin to ovaries, are organs that produce lipid hormones.
Significantly high TOS and OSI levels in tissue homogenates and blood serums of Omegaven Group seem like an unexpected finding in rough. But it might be due to high dose omega 3 exposure in this group which might also have some adverse effects. Some studies demonstrated that high dose omega 3 exposure might cause oxidative stress especially without vitamin E supplementation [24]. Ultrastructural comparisons reveal that Procarbazine adversely affects nearly all organelles. Lipid droplets, multivesicular structures, basal membrane thickening, and eosinophils are significant components of ultrastructural pathology. Omegaven also improves the ultrastructural results. In a 2015 study, investigators assessed the ultrastructural effects of the insecticide methyl parathion on ovaries. Lipid droplets and vacuoles were also seen in the ovaries [25]. This herbicide has been proven to cause infertility in male buffaloes by impairing sperm function [26]. Ovarian tissue atresia may exhibit characteristic atretic features regardless of the underlying cause. Diverse etiologies result in analogous ultrastructural data, suggesting that they may be viewed as a singular stage of atresia, wherein alterations in the ovarian microenvironment activate comparable processes.
Underlying mechanisms how alkylating agents impair ovarian reserve are not clarified enough. A recent review summarized that chemotherapy adversely affects the ovaries through many pathophysiological mechanisms, including inflammation, apoptosis, vascular alterations, cytokines, and immune system responses [27]. However, the elucidated mechanisms cannot account for the at least partially beneficial benefits of Gonadotropine Releasing Hormone (GnRH) analogs on women receiving chemotherapy [28]. Because GnRH analogs are not anti‐inflammatory, anti‐oxidant, or anti‐apoptotic drugs. Research presents contradictory findings on the impact of GnRH analogs on chemotherapy‐induced reduction of ovarian reserve [29]. Chemotherapy‐induced reduction in ovarian reserve may have broader mechanisms than inflammation, apoptosis, and oxidative stress.
Inflammation, oxidative stress, and apoptosis have been identified in the Procarbazine‐treated group in the current study also. Interestingly, electron microscopic inspection revealed the presence of eosinophils in the tissue of the Procarbazine group. The current study's results indicate that serum estradiol levels were significantly elevated in the Procarbazine group compared to the Procarbazine + Omegaven group. Although not statistically significant, tissue estradiol levels in the Procarbazine group were greater than those of all other groups. This finding must be evaluated with caution because the rats were not evaluated for cycle synchronization at the end of the experiment. A hyperestrogenic milieu could provoke an enhanced immune response and accelerated atresia of ovarian tissue [30]. A study on a rat model of airway inflammation induced by house dust mites revealed that estradiol augmented eosinophilia in lung tissue [31]. In conclusion, we observed beneficial effects of Omegaven on ovarian reserve in female rats subjected to Procarbazine. Unexpected findings of the current study like eosinophil increment in the damaged tissue, Estrodiol levels, increased some oxidative stress parameters in the group only treated with Omegaven must also be further investigated.
The mechanisms behind Omegaven's protective effects on ovarian function in Procarbazine‐exposed rats and other alkylating drugs that exert deleterious effects on the ovaries require elucidation in subsequent investigations. Although a major limitation of our study is the method used in follicle count, they were supported and strengthened by biochemical findings. Further investigations are required to assess the clinical application of Omegaven in preserving ovarian reserve in women subjected to chemotherapy. With the advancement of research, it may soon be feasible for young cancer survivors to have biological children and lead a healthy life with their endogenous estrogen.
Funding
This project received assistance from The Scientific and Technological Research Council of Turkey, TUBITAK [grant number: 223S989].
Disclosure
The authors have nothing to report.
Ethics Statement
Cukurova University, Animal Studies Local Ethical Committee, Meeting Number: 7, Date: 7/10/22, Decision Number: 2.
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Data S1: Supporting Information.
Acknowledgments
Gratitude is extended to our adviser, Prof. Dr. Sait Polat, from Cukurova University, Faculty of Medicine, Department of Histology and Embryology. Gratitude is extended to the project's scholars: Ayse Ceren Timur, Asli Sena Celiker, Meryemay Ogulcan, İlkay Calisir, Mehmet Yagiz Capanoglu, and Ahmet Riza Erzurumlu. During the preparation of this work the author(s) used Quillbot Premium in order to paraphrase, grammar check, and humanize the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.
Data Availability Statement
The data that supports the findings of this study are available in the Supporting Information of this article.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data S1: Supporting Information.
Data Availability Statement
The data that supports the findings of this study are available in the Supporting Information of this article.