PRECIS: Hyperbaric oxygen therapy in premature ovarian failure.

Introduction

The total number of immature primordial follicles found in the ovaries is known as the ovarian reserve. Estimation of the ovarian reserve is accepted as a basic marker of fertility With a process known as “activation,” the rate at which primordial follicles grow is an important point affecting fertility. From a total of approximately 1-2 million primordial follicles at birth, only approximately 400 will mature into primary oocytes with the capability for ovulation and fertilization in the reproductive years of a woman. The vast majority are victims of atresia.

When approximately 1.000 primordial follicles remain, this causes fertility to stop and menopause to start⁽¹⁾. Information about the renewal of primordial follicles related to the depletion of the ovarian reserve has started to be discussed with the emergence of evidence related to the identification of oogonial stem cells, which can produce new oocytes in mouse ovaries⁽²⁾, and their presence in human ovaries⁽³⁾. However, there is limited knowledge about the underlying molecules and biochemical processes of follicular activation in humans. Moreover, exposure to exogenous factors such as stress, radiation, anti-neoplastic agents, and cigarette smoke cause the formation of reactive oxgen species, which can develop in the body under physiological conditions. Oxidative stress occurs because of an increase in the oxidant level and/or a decrease in the antioxidant level in the body. Oxidative stress causes damage to cellular mitochondria, nucleus, and membranes. Free radicals cause irreversible damage in the organism with the effect of damage to deoxyribonucleic acid (DNA). It is thought that reactive oxygen species (ROS) plays an important regulatory role in folliculogenesis, oocyte maturation, and luteolysis.

Chemotherapeutic agents have a negative effect on the reproductive potential of young women, but the mechanisms through which this occurs are still unclear. Cyclophosphamide (CYP) is used to treat several cancer types, primarily leukemia and lymphoma, which are the most frequently seen cancers in adolescent females⁽⁴⁾. Alkylating agents, especially CYP, are extremely toxic for the gonads because the toxicities are independent of cell proliferation⁽⁵⁾. CYP directly damages DNA, induces follicular apoptosis, and produces ROS, which damage ovarian cells⁽⁶⁾. CYP also leads to over-activation of primordial follicles by actvating the P13K/AKT/mTOR pathway⁽⁷⁾. This is accepted as a factor that is effective in the early reduction of follicles.

Premature ovarian insufficiency (POI), which is a cause of female infertility, is defined as failure in ovarian functions for a period of longer than 4 months before the age of 40 years, and is characterized by amenorrhea, elevated gonadotropin levels, and hypooestrogenism⁽⁸⁾. This condition affects 0.3-1% of women⁽⁹⁾. POI can occur with the depletion of the ovarian reserve associated with acceleration in the rate of primordial follicle activation⁽¹⁰⁾, primordial follicle loss⁽¹¹⁾, or iatrogenic causes such as chemotherapeutic agents⁽¹²⁾. These patients experience physiological symptoms associated with hypooestrogenism, infertility, and a series of psychological problems⁽¹³⁾. Simultaneously, there is an increased risk of cardiovascular disease, osteoporosis, urogenital atrophy, and neurodegenerative disease in these patients⁽¹⁴⁾. Therefore, the prevention of POI is important to protect women against infertility, and other systemic problems brought about by early menopause.

Hyperbaric oxygen therapy (HBO) is a treatment method in which 100% oxygen is breathed by exposure to increasing atmospheric pressure. Recently, it has been used as an effective adjuvant treatment method for treating several different pathologies. The oxygen present can re-oxygenate areas where hypoxia or hypoperfusion has occurred.

This study aimed to determine the possible effects that could formed after HBO therapy in rats applied with CYP to create a premature ovarian failure model, by examining ovarian histology and the serum parameters of Anti-müllerian hormone (AMH), total antioxidant status (TAS), and total oxidant status (TOS).

Materials and Methods

This study was approved by the Erciyes University Animal Experiments Ethic Committee (no: 21/168; date: 07.07.2021). The work was funded by the Erciyes University Scientific Research Projects Coordination Unit (TSA- 2022- 11550).

The study material comprised 18 adult, female Wistar rats, aged 10-12 weeks, each weighing 200±20 gr. The rats were kept under observation for 3 days, for adaptation, for signs of any health problems. Throughout the study, the animals were kept under stable conditions of 22 °C environmental temperature, 30-70% humidity, a 12-hour light/dark cycle, with food of dry pellets and tap water.

The rats were separated into 3 groups of 6 animals in each. The control group was named group 1, the CYP group, group 2, and the CYP+HBO group, group 3. The control group rats were administered 1 mL/kg physiological saline intraperitoneally (IP) for 15 days. The rats in group 2 and group 3 were injected IP with 200 mg/kg CYP on day 1, based on the protocol in the study by Melekoglu et al.⁽¹⁵⁾ followed by 8 mg/kg/day for 14 days to create POI. From the 15^th day onwards, the rats in the CYP+HBO group were placed in a hyperbaric cabin and exposed to 100% oxygen at 2.4 atm pressure for one h, and were then returned to their cages at the end of the hour.

For the HBO therapy, the cabin was filled with 100% oxygen until the pressure reached 2.4 atmosphere at the rate of 0.1 bar/min in 15 min. At the end of 1 h of treatment, the air in the cabin was safely released until it fell to a normal pressure of 1 atmosphere at a rate of 0.1 bar/min. This procedure was continued at the same duration and pressure for 14 days.

On day 30, all the rats in all groups were administered general anesthesia of xylazine (10 mg/kg live weight, IP) and ketamine (60 mg ketamine hydrochloride/kg live weight, IP). First, an intracardiac blood sample of approximately 5 cc was taken. The serum was separated by centrifugation at 3.000 rpm for up to 10 min. While the animals were under deep anesthesia, a 2 cm skin incision was made over the linea alba in the abdominal region. The subcutaneous connective tissue and abdominal muscles were opened, and oophorectomy was performed by dissecting the left and right ovaries. The rats were then sacrificed with the cervical dislocation method.

Ovarian tissues were fixed in 10% formaldehyde solution for 48 h. The tissues were washed under running tap water, and then passed through increasing graded alcohol series. After being made transparent with xylene, the tissues were embedded in paraffin blocks. Slices 5 µm in thickness were cut from the paraffin blocks, stained with Hematoxylin and Eosin and Masson Trichrome, and then blinded examined under an Olympus BX51 microscope. Evaluations were made according to the determined criteria. The germinal epithelium and tunica albuginea were measured using ImageJ software.

Determination of TAS and TOS Levels

Serum TAS and TOS levels were determined using commercial Rel Assay kits (Rel Assay Kit Diagnostics, Turkey). Trolox was used as a calibrator for TAS tests, and the results were expressed in mmol Trolox equivalent/L. Hydrogen peroxide was used as a calibrator for TOS tests and the results were expressed in mmol H₂O₂ equivalent/L.

ELISA Analysis of AMH Levels

Serum samples were collected by centrifugation (5000 rpm for 10 min) and stored at -80 °C. The procedure was performed with an AMH ELISA Kit (AFG Bioscience, USA) following the manufacturer’s instructions. Optical density (OD) values were measured at 450 nm using a spectrophotometer (BioTek, Synergy H, TX, USA). The concentrations of AMH in the serum samples were determined by comparing the OD values of the samples to the standard curve.

Statistical Analysis

The SPSS 22 software was used for statistical analysis. Statistical differences between groups were tested using One-Way ANOVA, and comparisons between the control and treated groups were performed using an unpaired Student’s t-test. The data werre presented as mean ± standard error of the mean values. A value of p<0.05 was accepted as statistically significant.

Results

Serial slices (5 µm) were taken from the ovarian tissues obtained. One in five of the slices were stained, and follicles were counted. A statistically significant decrease was determined in the primordial and primary follicle counts in group 2 compared to group 1 (p<0.05). A statistically significant increase was determined in the primordial and primary follicle counts in group 3 compared to group 2 (p<0.05). When the groups were evaluated according to pre-antral and secondary follicle counts, the only statistically significant difference was between group 1 and group 2 (p<0.05). A statistically significant difference was determined between group 2 and both groups 1 and 3 with respect to secondary follicle count (p<0.05) (Table 1).

When the groups were evaluated with respect to the germinal epithelial thickness, the difference between group 1 and group 3 was statistically significant (p>0.05). An increase occurred in the mean germinal epithelial thickness of group 2, and this increase was determined to be statistically significant compared to groups 1 and 3 (p<0.05) (Table 1). In the evaluation of tunica albuginea thickness, a statistically significant difference was determined between all groups (p<0.05) (Table 1).

In group 1, tunica albuginea formed of fibrous connective tissue fibers and cells was observed below the germinal epithelium. Below the tunica albuginea was the cortex section, which was in the more peripheral part and contained various numbers and types of follicles, and the medulla, containing a rich vascular bed and with a loose connective tissue structure in the inner section. The germinal epithelium in group 3 was similar to that in group 1, but the tunica albuginea layer was seen to be thicker than in the control group. In group 2, in the whole tunica albuginea layer, including the cortex, there was more blood vessels and wider diameter of the vessels. In the primordial, primary, pre-antral, secondary, and tertiary follicles, the oocytes and the granulosa cells surrounding the oocytes were irregular. There was also observed to be an evident increase in the number of atretic follicles in group 2. The connective tissue sheath (theca) surrounding the follicle, and which differed from the stromal cells that began to be observed from the primary follicles, was enlarged and irregular. The granulosa lutein cells in the corpus luteum, which develops after ovulation, were observed to be irregular in shape with large gaps between them. There was a significant increase in the number of blood vessels in the medulla layer, and widening of the blood vessel diameters could be significantly differentiated. The histopathological findings in group 3 were close to those in group 1.

The number of blood vessels was decreased and the diameters of the vessels were narrower. Fewer atretic follicles were observed. The theca layer of the connective tissue surrounding the follicles was more regular in shape (Figure 1).

When the serum AMH levels of the groups were examined, the serum AMH levels in group 2 were seen to be significantly decreased compared with both group 1 and group 3 (p<0.05) (Table 2). The highest mean serum AMH level was determined in group 3 (8.83±0.87 ng/mL) but this was not at a statistically significant level compared to the control group (8.79±1.37 ng/mL).

These results showed that CYP lowered the AMH level significantly, but the application of HBO therapy to these patients with CYP could prevent a fall in serum AMH levels.

When the TOS values were examined, there was determined to be statistically significantly higher oxidant status in group 2 than in group 1 (7.02±2.29 vs. 3.85±0.62, p=0.0083) (Table 2). In group 3, the HBO therapy was seen to have statistically significantly reduced the oxidant status (3.70±1.19, p=0.010). This status can be considered protective with respect to the ovarian follicles.

The TAS levels were determined to be statistically significantly higher in group 1 than in group 2 (1.10±0.11 vs. 0.79±0.12, p<0.001) (Table 2). In group 3, HBO was determined to have statistically increased the TAS values (1.27±0.22, p=0.0008).

Discussion

In this study, the effect of HBO therapy was examined on the ovarian reserve in a rat model of ovarian failure created with CYP. The results of the study showed that there was a significant decrease in ovarian reserve with CYP, then with HBO therapy after CYP, and a statistically significant increase was determined histopathologically in the primordial and primary follicle counts in these rats. From an examination of the literature, it can be seen that the current study is the first to have evaluated the effects of HBO in preventing CYP-related ovarian damage, and to have shown that HBO has a therapeutic effect against ovarian failure of CYP origin.

Chemotherapeutic agents have a negative effect on the reproductive potential of young women, but the mechanisms through which this occurs are still unclear. Alkylating agents, especially CYP, are toxic for the gonads because they are independent of cell proliferation⁽⁵⁾. CYP is used to treat several cancer types, primarily leukemia and lymphoma, which are the most frequently seen cancers in adolescent females⁽⁴⁾. CYP directly damages DNA, induces follicular apoptosis, and produces ROS, which damage ovarian cells⁽⁶⁾. CYP also leads to over-activation of primordial follicles by activating the P13K/AKT/mTOR pathway⁽⁷⁾.

Morgan et al.⁽¹⁶⁾ concluded that acute loss of the growing follicle population during chemotherapy results in increased intake of primordial follicles to the growing follicle pool. This damage shows two clinical phenotype stages: 1) amenorrhea, which is generally reversible and usually occurs a short time after chemotherapy, and 2) early menopause, which is generally irreversible and related to over-activation of primordial follicles by chemotherapy. In this study, a statistically significant decrease was determined in the number of primordial and primary follicles in the group administered CYP compared with the control group.

There is a great deal of evidence linking exposure to CYP with follicle atresia and granulosa cell apoptosis^(17,18). It has also been reported that inflammation and vessel expansion with CYP create secondary vascular damage⁽¹⁹⁾. In this study, a thickened tunica albuginea, and increased vessel structure and vessel expansion in the germinal matrix were determined. This finding of increased vascularization and expansion is consistent with the literature. Considering that venous congestion is thought to cause ovarian insufficiency, free oxygen radicals and inflammatory cytokines can be considered to accumulate more intensively in the ovarian tissue.

When the literature is examined, it can be seen that there has been an investigation of potential protective agents (eg., Curcumin and capsaicin, Tamoxifen, Sphingosine1-phosphate) against the direct loss or accelerated activation of primordial follicles, increasing atresia of growing follicles, and damage to the vascular system in the ovaries, caused by CYP^(15,20,21). In the study by Melekoğlu et al.⁽¹⁵⁾, it was revealed that CYP causes premature ovarian failure, and it was concluded in this study that Curcumin (CRC) and Capsaicin (CPS) can be used to prevent premature ovarian failure in rats. It was observed that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels decreased in rats receiving CRC and CPS, and simultaneously, amh levels increased with estrogen.

The results of the current study clearly showed that CYP reduced the number of primordial and primary follicles in rat ovaries. The numbers of pre-antral and secondary follicles in the rats administered CYP were also observed to be lower compared with the control group and the rats that received HBO.

The ovaries express the vascularity of primordial and pre-antral follicles from the stromal blood vessels. Additionally, the growth of primary follicles leads to the development of the vascular network in the theca layer. Embryos developing from oocytes obtained from well -vascularised follicles have a higher implantation rate than embryos obtained from oocytes developing from follicles with poor vascularity⁽²²⁾.

The positive effects of HBO on wound healing have been documented in animal experiments. There are many studies showing that HBO increases collagen production, supports angiogenesis and leads to the development of the vascular network causing the formation of new capillaries by inducing the release of stem cells from the bone marrow into circulation, thereby promoting mechanisms that trigger regeneration and healing^(23,24).

HBO therapy triggers the angiogenesis mechanism by providing the oxygen needed and with increased release of several cytokines such as vascular endothelial growth factor⁽²⁵⁾. Angiogenesis is of critical importance for follicular development, oocyte quality, and early embro development. This showed that adjuvant HBO therapy after chemotherapy could regulate ovarian function and hormone levels. Mitrović et al.⁽²⁶⁾ showed that HBO therapy could be a potential treatment option for infertility by increasing endometrial receptivity through a change in blood vessel resistance. In this study, HBO therapy was seen to correct the abnormal expansions in the blood vessels created by CYP, reduced congestion, and provided a normal vascular structure close to that of the control group. This correction formed in the vascular structure suggests that follicle loss is prevented by reducing oxidative stress and inflammatory cytokines in the ovary.

In addition to reducing follicle loss, that HBO showed an effect on serum AMH levels was among the main aims of this study. Yu et al.⁽²⁷⁾ examined the effect of HBO therapy on ovarian function following cystectomy. A total of 60 patients with ovarian cysts were treated with laparoscopic ovarian cystectomy. HBO was added to patients in the observation group in addition to the treatment in the control group. The AMH, FSH, LH, estradiol (E2), and antral follicle count (AFC) serum levels were detected in both groups before the operation and at the first and third menstrual cycles postoperatively to evaluate ovarian function. After the operation, AMH, E2, and AFC serum levels in the observation group were significantly higher than in the control group. FSH and LH serum levels were significantly lower than in the control group, and the differences were statistically significant⁽²⁷⁾. In a study of 4 patients by Pineda et al.⁽²⁸⁾, the effect of HBO was examined on serum AMH levels. In 2 of the 4 patients, the serum AMH level showed an increase of 40% and 116%. In one patient, no change was observed in the serum AMH level, and the other patient became pregnant during the HBO therapy. When the serun AMH levels were examined in the current study, the AMH levels were found to be significantly higher in group 3 than in group 2, which was consistent with the literature⁽²⁸⁾.

Although some studies have shown that HBO-induced oxidative damage^(29,30), several others have stated that HBO has a protective effect against oxidative damage^(31,32). The debate is ongoing on whether HBO acts as an oxidant promoter or as an antioxidant agent. It has been shown that ROS and mitochondrial DNA (mtDNA) affect cellular aging in the human body, including the female reproductive system. More importantly, studies have suggested that ovarian aging could be negatively affected by excessive ROS⁽³³⁾. Van Blerkom et al.⁽³⁴⁾ showed the importance of oxygen in oocyte meiosis. It was reported in that study that a decrease in oxygen content in the ovarian follicular fluid in humans is associated with an increase in abnormalities in the organization of chromosomes on the metaphase spindle, and it was emphasized that a sufficient oxygen source is necessary to allow the acceleration of chromosomes and normal oocyte maturation^(34,35). In this study, TOS was seen to be increased in group 2 compared with group 1, and TAS was statistically significantly higher in group 3 than in group 2. These results suggest that follicle loss in the ovaries was reduced and the negative effects of CYP were prevented with HBO therapy.

Study Limitations

The strength of our study is that it evaluated both tissue oxidative stress markers, ovarian reserve markers, and histopathological parameters together. The limitation of our study is that this study is an animal experiment, and further clinical studies should be done to show its effect on humans.

Conclusion

CYP negatively affects the ovarian reserve and causes severe follicle loss. HBO therapy could be an alternative treatment to minimize the effect on ovarian follicle loss of these alkylating agents, which must be used for treating tumors that commonly occur in young females of reproductive age. Although different pressures and durations of HBO were not applied in this study, the dose applied suggests that HBO could be protective for the ovaries. There is a need for further clinical trials to provide more clarification on this subject.

Ethics

Ethics Committee Approval: This study was approved by the Erciyes University Animal Experiments Ethic Committee (no: 21/168; date: 07.07.2021).

Informed Consent: Not necessary.

Peer-review: Externally peer-reviewed.

Authorship Contributions

Surgical and Medical Practices: F.Ç., M.A.B., A.C., M.E., Concept: F.Ç., M.A.B., E.M.A., Design: F.Ç., M.A.B., M.D., Data Collection or Processing: E.B., A.C., B.Y., Analysis or Interpretation: E.B., A.C., B.Y., Literature Search: F.Ç., M.A.B., M.D., E.M.A., Writing: F.Ç., M.A.B., M.D., E.B., A.C., E.M.A.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The work was funded by the Erciyes University Scientific Research Projects Coordination Unit (TSA- 2022- 11550).