Objectives: Information is scarce on levels of kisspeptin-1 and the kisspeptin-1 receptor in females after ovarian transplant. In this study, our aim was to explore serum estradiol, anti-Müllerian hormone, kisspeptin-1, and kisspeptin receptor levels, along with kisspeptin-1-positive cell density, in ovaries from rats after ovarian transplant.

Materials and Methods: For this study, 28 female Sprague Dawley rats were divided into 4 groups, with sham surgery performed on rats in group 1 (control group). Group 2 rats had ovaries transplanted under the peritoneum, and group 3 rats had their own ovaries transplanted subcutaneously. Group 4 rats were maintained in an estrous state. Serum anti-Müllerian hormone, kisspeptin-1, estradiol, and ovarian kisspeptin receptor levels were determined using commercial enzyme-linked immunosorbent assay kits. Kisspeptin-1-positive cell densities in the ovaries were determined immunohistochemically. The ovaries were also examined histopathologically.

Results: Our statistical analyses showed that levels of kisspeptin receptors in the ovaries were lowest in the subcutaneously transplanted group (group 3; 628.57 ± 35.69 pg/mL). The highest serum anti-Müllerian hormone levels were found in the estrous group (group 4; 16.91 ± 2.12 ng/mL). Differences between groups in terms of serum kisspeptin-1 and estradiol concentrations were not statistically significant.

Conclusions: Our findings suggest that, in rats, results were better in the peritoneum transplant group than in the subcutaneous transplant group. We also found that serum anti-Müllerian hormone levels were lower in the transplant groups than in the estrous group, although levels were not completely decreased to zero. These results support the finding that ovarian activities continue after transplant.

Key words : Animal model, Estradiol, Infertility

Introduction

Anti-Müllerian hormone (AMH) inhibits fetal development by blocking the development of the Müllerian ducts in males.^1,2 Anti-Müllerian hormone, which is thought to show its effect only in repro­ductive organs, plays an important role in the development of the male fetus in intrauterine life. Presence of AMH enables regression of the Müllerian ducts, which promote the development of the uterus, fallopian tubes, and the upper part of the vagina, thus allowing normal development of the male reproductive system.³

Kisspeptin is synthesized in the testis, ovary, pancreas, intestines, and most intensively in the placenta, together with the central nervous system.⁴ The G-protein coupled GPR54 receptors are distributed diffusely in the central nervous system and have been shown to be located on gonadotropin-releasing hormone neurons in the hypothalamus. Kisspeptin directly stimulates gonadotropin-releasing hormones through GPR54.⁵ Previous experimental animal models have demonstrated that central or peripheral administration of this peptide strongly stimulates the hypothalamic-hypophyseal-gonadal axis and increases gonadotropin levels.^6,7 In contrast, the infusion of the kisspeptin antibody in female rats completely stopped the estrous cycle.⁸ The expression of the kisspeptin-1 gene (KISS1) in the hypothalamus is modulated by gonadal steroids.⁷

Ovarian transplant is performed to solve infertility problems caused by various ovarian disorders and also to maintain endangered species, to investigate follicular dynamics and folliculogenesis, to reduce the adverse effects of chemotherapy on ovaries in women receiving cancer treatment, and to prevent the complications of oophorectomy operations in carnivores.9,10 The entire or part of the ovary can be transplanted fresh or can be frozen and stored over a period of time.

In this experimental study, our aim was to compare serum estradiol, AMH, kisspeptin receptor, and KiSS-1 protein levels, as well as KiSS-1-positive cell density, in ovaries in the ovary transplanted rat model.

Materials and Methods

Experimental animals
In this study, 28 female Sprague Dawley rats aged 2 to 4 months old and weighing 200 to 250 g were used. The rats were obtained from the Center for Experimental Research at Fırat University (Elazig, Turkey). During the study, the rats were caged in groups and subjected to a 12:12-hour dark-light cycle. Rats were maintained on ad libitum food and water. The experimental rats included in the study had a regular sexual cycle that was confirmed by detecting that they had at least 4 regular cycles in successive vaginal irrigations. This study was approved by the Fırat University Experimental Animals Local Ethics Committee (30.11.2016-2016/21).

Application groups
Rats were grouped as follows. Group 1 (n = 7) was selected as the control or sham group, and rats underwent sham operation. Group 2 rats (n = 7) underwent bilateral oophorectomy, and removed ovaries were transplanted to the same animal under the peritoneum bilaterally on the abdominal wall (peritoneum group). Group 3 rats (n = 7) had removed ovaries transplanted subcutaneously into the inguinal pelvic region of the same animal after bilateral oophorectomy (subcutaneous group).11 Group 4 consisted of rats (n = 7) that were found to be at estrus (estrus group).

For surgical procedures, first, anesthesia was administered with xylazine (10 mg/kg intra­muscularly) and ketamine (90 mg/kg intra­muscularly) and then ovaries were subsequently transplanted. The operations were carried out in the Fırat University Experimental Research Center’s laboratories.

Vaginal irrigation
Vaginal irrigation was performed as described by Risvanli and associates.¹² Irrigations were made with sterile distilled water using a rubber pail and pipetter. The liquid obtained after irrigation was placed on the slide and examined under a microscope at ×40 magnification. The densities of the cell types in the preparations were rated as +, ++, and +++. The rats with +++ superficial cell density were considered to be in the estrus state.

Laboratory analyses
Blood and tissue specimens of rats were taken as soon as estrus was detected in the estrus group and 1 month after transplant in the other rat groups after decapitation. Sera of decapitated rats were stored at -20°C until measurements were carried out.

Measurement of serum anti-Müllerian hormone levels
Levels of AMH in blood sera were determined by an enzyme-linked immunosorbent assay (ELISA) reader (BioTek Instruments, Winooski, VT, USA) using a commercial ELISA kit (YLA0062RA; YL Biont, Shanghai, China) (sensitivity of 0.051 ng/mL).

Measurement of serum kisspeptin-1 levels
Kisspeptin-1 levels in blood sera were determined by ELISA readers (BioTek Instruments) using a com­mercial ELISA kit (EA0873Ra; Li StarFish, Cernusco sul Naviglio, Italy) (sensitivity of 2.49 pg/mL).

Measurement of ovarian kisspeptin receptor levels
Levels of kisspeptin receptor in rat ovaries were determined by an ELISA reader (BioTek Instruments) using a commercial ELISA kit (YLA1199RA; YL Biont Shanghai) (sensitivity of 4.98 pg/mL). Before measurements were carried out, the ovaries were homogenized as described below.

Homogenization
Kisspeptin receptors have been detected in ovary-related tissues only. Tissues with fibrosis were removed from the ovaries and frozen at -20°C. After they were removed from the freezer, homogenization was performed in ice molds at 4°C with 0.01 M phosphate-buffered saline (PBS; pH 7.4) at 1:10 dilution. After homogenization, the supernatant parts were separated by centrifugation at 3000 revolutions/min for 10 minutes and used in further analyses.

Measurement of serum estradiol levels
Estradiol levels in blood sera were determined by an ELISA reader (BioTeaters) using a commercial ELISA kit (CK-E10719; Eastbiopharm, Hangzhou, China) (sensitivity of 1.54 ng/L).

Histopathologic analyses
The left ovary of each rat in the experimental groups was removed and fixed in 10% neutral formalin solution. Paraffin blocks were prepared after routine processes. Five sections (5 μm thick) were taken from paraffin blocks for each sample and stained with hematoxylin and eosin and examined under light microscope. Total counts of secondary follicles, inverted follicles, atretic follicles, and corpus luteum were evaluated from these 5 sections prepared for each rat. In addition, histopathologic changes (inflammation, fibrosis, fat infiltration, pigmentation, follicular cysts, and luteinized cysts) were recorded for each section.

Immunohistochemical analyses
To determine the KiSS-1-positive cell distribution in the ovaries, paraffin sections were immuno­histochemically stained with the avidin-biotin complex method using mouse- and rabbit-specific horseradish peroxidase/3-amino-9-ethylcarbazole detection kits. The staining was carried out according to the manufacturer’s protocol. A rabbit anti-KiSS-1/kisspeptin polyclonal antibody kit (bs-0749R; Bioss Antibodies, Woburn, MA, USA) was used as the primary antibody. Sections taken from paraffin blocks (5 μm thick) were put into poly-L-lysine-coated slides. After sections were deparaffinized with xylol and rehydrated in graded alcohol solutions, tissue samples were incubated in a microwave oven for 20 minutes in a citrate buffer solution for antigen retrieval. To deactivate endogenous peroxidase, sections were incubated for 10 minutes in a 3% hydrogen peroxide solution. The tissues were washed with PBS solution 3 times at 5-minute intervals, and then tissue samples were incubated for 10 minutes in normal serum. Without washing, the tissues were incubated with the primary antibody at 1/200 dilution at 4ºC overnight. The tissues were then washed in PBS and incubated for 10 minutes in a peroxidase-conjugated biotinylated anti-rabbit solution. The tissues were again washed 3 times in PBS and incubated for 30 minutes with horseradish peroxidase conjugate for 30 minutes followed by treatment with 3-amino-9-ethylcarbazole chromogen. Mayer hematoxylin was used for counterstaining. Tissue samples were glued with mounting medium and evaluated with a light microscope.

Tissues were scored as follows: - (no staining), + (light staining), ++ (moderate staining), and +++ (intensive staining). For negative control, a nonimmunized serum was used instead of the primer antibody.

Statistical analyses
For this study, 4 independent treatment groups were formed. The total sample size (rat number) for all treatment groups was determined to be 76 according to effect size (0.40), alpha (0.05), and power (0.80) with G-power test (that is, at least 19 rats were required for each group). However, because rats would need to be killed during the research process, the local ethics committee recommended that the rat number be lowered to 7 animals per group according to 3R rules (replacement, reduction, refinement), as also used in some similar studies. Therefore, we determined that 7 rats for each group for this study were adequate.

For statistical analyses of data, descriptive statistics of the characteristics were calculated first. Obtained data were examined for each group to see whether they met the parametric test assumptions and showed normal distribution. The Kruskal-Wallis analysis of variance, a nonparametric measure of the one-way analysis of variance, was used for com­parisons of examined characteristics among groups. This analysis was also done because we had a lower number of rats per group, data did not show normal distribution, and the experimental groups were independent groups. The adjusted Bonferroni Mann-Whitney U test was applied as a post hoc test for the variables, with significantly differences among groups determined with the Kruskal-Wallis test. P values < .01 were considered as statistically significant. We used SPSS version 22.0 software (SPSS Inc., Chicago, IL, USA) for statistical analyses.

Results

Laboratory analyses
Kisspeptin receptor levels in the ovarian tissue were found to be lowest in the subcutaneous group (628.57 ± 35.69 pg/mL), in which the ovaries were transplanted subcutaneously (P < .05). Although we observed numeric differences between the peritoneum and subcutaneous groups with regard to kisspeptin receptor levels, these results were not statistically significant. Serum AMH levels were highest in the estrus group (16.91 ± 2.12 ng/mL; P < .05), and serum KiSS-1 and estradiol concen­trations were not statistically different between groups (P > .05) (Table 1).

Histopathologic analyses
In the sham group, histopathologic examination showed that none of the rats had inflammation, fibrosis, or cysts, and only 2 rats had pigmentation.

In the peritoneum group, fibrosis was detected in all rats, inflammation was observed in the ovaries of 6 rats (Figure 1A), and fat infiltration was observed in the ovaries of 6 rats (Figure 1B). Lipid cells were burst in some places, and lymphoplasmacytic and macrophage infiltrations with giant cells (Figure 1C) and connective tissue proliferation were observed in their surroundings. In addition, pigmentation was detected in 4 rats, follicular cysts in 3 rats, and surface inclusion cysts in 1 rat.

In the subcutaneous group, inflammation was observed in the ovaries of 4 rats, fibrosis in 6 rats, fat infiltration in 6 rats, and follicular cysts in 3 rats (Figure 1D). One rat showed luteal cysts (Figure 1E), and pigmentation in ovaries occurred in all rats (Figure 1F).

None of the rats in the estrus group had any histopathologic findings such as inflammation, fibrosis, fat infiltration, follicular cysts, luteal cysts, or pigmentation.

The histologic findings of the physiological structures on ovaries are summarized in Table 2. Statistically significant differences were found among rat groups in terms of other parameters, except for atretic follicle count (P < .001). None of the ovaries obtained from the peritoneal and subcutaneous groups included secondary folliculi. Tertiary follicle counts were lowest in the peritoneum (1.57 ± 0.65; P < .001) and in the subcutaneous groups (1.71 ± 0.61; P = .049). The corpus luteum count was also found to be lowest in the peritoneum (2.57 ± 1.11; P < .001) and in the subcutaneous groups (4.71 ± 1.17; P < .001).

Immunohistochemical analyses
The presence of kisspeptin was detected by red-colored cytoplasmic staining in immunoreactive cells. No staining was observed in sections using nonimmunized serum instead of primer antibody. Kisspeptin was found extensively in granulosa cells of atretic follicles (Figure 2A), in interstitial cells (Figure 2B), and in luteal cells of the corpus luteum, as shown by immunopositive staining. Strong positive staining was observed in the oocyte cytoplasm layer, whereas weak staining was observed in the oocytes (Figure 2A). No positive staining was observed in the granulosa cells of primordial, primary, secondary, and tertiary follicles in all experimental group rats. The most intense kisspeptin immunoreactivity was noted in corpus luteum in the early phase. Compared with the sham and estrus groups, the intensity of staining in the corpus luteum decreased in the peritoneum and subcutaneous groups (Figure 2, C and D), whereas the intensity of staining in interstitial cells was increased. The immunohistochemical localization and intensity of the kisspeptin in the ovaries are presented according to experimental group in Table 3.

Discussion

There are limited publications investigating the relationship between ovarian transplant and serum AMH concentrations. It has been reported that ovary functions and serum AMH concentrations return to normal in women who have had their own frozen ovarian tissue reimplanted.¹³ In another case report, a woman with serum AMH concentration of 0.6 to 1.8 g/L after ovarian transplant had a slight increase in postoperative serum AMH level compared with the low levels demonstrated preoperatively.¹⁴ In another study,¹⁵ serum AMH concentrations were reported to be low or immeasurable in all women who underwent ovarian transplant, depending on the postoperative duration. In addition, in the same study, higher serum AMH levels were detected in women who received freshly transplanted ovaries versus those who received frozen and thawed ovaries. It has also been suggested that the number of antral follicles detected histologically is correlated with serum AMH concentrations, although this may vary with age.¹⁶

In a study in which fresh or frozen ovarian tissue from women had been transplanted in severe combined immunodeficient mice,¹⁷ AMH expression was seen up to 28 weeks after transplant. Thus, this hormone was suggested to be a factor of prolonged survival of ovarian tissues. Silber and colleagues¹⁸ found that women who received fresh or frozen ovarian allografts or autotransplant had serum AMH concentrations above normal until postoperative day 170, which then dropped below the normal level at around postoperative day 240 and remained at this low level.

It was also reported that the use of serum AMH concentrations as a marker of ovarian reserves may sometimes be misleading. For example, women with low serum AMH concentrations after ovarian transplant have been reported to become pregnant.¹⁵ In this context, serum AMH concentrations alone cannot be the determinant of fertility. It is suggested that serum inhibin B and follicle-stimulating hormone concentrations are also essential for fertility together with this hormone.^16,19 According to the results of these studies, it can be concluded that the normalization of serum AMH concentrations after ovarian transplant depends on the activity of transplanted ovarian tissues, the technique used, and the postoperative duration. In our study, serum AMH levels were 2.74 ± 0.28 and 2.96 ± 0.21 ng/mL in the subcutaneous and peritoneal groups, res­pectively, with levels shown to be highest in the estrus group. These concentrations, which were collected in the first month posttransplant, are similar to the decrease in serum AMH concentration in the early period after transplant shown in women. Our findings regarding results at the first month after transplant are consistent with previous studies, which showed a decrease in serum AMH concentration in women who are in the early period after ovarian transplant.¹⁵

In a study that assessed plasma levels of AMH as a marker of the preantral follicle population,²⁰ kisspeptin was shown to increase plasma AMH levels in 6- and 10-month-old rats. In the same study, it was suggested that application of kisspeptin to rats could increase the number of small antral follicles, which in turn could increase the concentration of AMH known to be secreted from the secondary and small antral follicles.

According to information obtained from previous studies on various species, including histopathologic examination of transplanted ovaries after removal from the site where they were transplanted, increased vascular density can be an indication of vascularization. Although inflammation and fibrosis were found in ovaries, antral as well as primary and secondary follicles were detected. These studies also reported that, although follicle development continues after autotransplant, there is a reduction of up to 50% in the number of follicles.^21-23 In our study, we detected a number of pathologic conditions, including inflammation, pigmentation, follicular cysts, fat infiltration, and fibrosis, during histopathologic examination of the transplanted ovaries of the rats in the peritoneum and subcutaneous groups. Our observations were consistent with those shown in previous studies. Our histologic examination of the physiological structures of ovaries showed statistically significant differences among all groups regarding all variables, except for atretic follicle counts (P < .001). None of the ovaries in the peritoneum and subcutaneous groups were found to have secondary follicles. Tertiary follicle counts were also lower in the peritoneum (1.57 ± 0.65; P < .001) and subcutaneous groups (1.71 ± 0.61; P = .049). The number of corpus luteum was also found to be lowest in the peritoneum (2.57 ± 1.11; P < .001) and subcutaneous groups (4.71 ± 1.17; P < .001).

Callejo and associates²³ found that estradiol was higher in rats that underwent bilateral oophorectomy and that had ovaries transplanted intraperitoneally and subcutaneously compared with the control group, whereas serum follicle-stimulating hormone concentrations remained unchanged for 6 months at a lower level compared with results in controls. The group concluded that this result originated from estradiol production of the transplanted ovarian tissues. Callejo and associates²⁴ also reported that follicle-stimulating hormone concentrations were higher after subcutaneous ovarian transplants, whereas estradiol levels were higher than those measured in the control group but lower than the normal level. In another study,²⁵ a trophic maturation was observed in vaginal smears gathered between 4 and 10 days after intraperitoneal and subcutaneous ovarian transplant following bilateral oophorectomy. The investigators observed atrophic findings in the control group and concluded that the transplant method does not affect the results. In our study, serum estradiol concentrations in the rat transplant groups were similar to those shown in our rat estrous and sham groups, which were used as the controls. These results suggest that subcutaneous or subperitoneal ovarian transplant does not affect estradiol concentrations and that the comparable serum estradiol levels in the control and transplant groups suggest that follicular activity of the ovaries is maintained.

To the best of our knowledge, there are no data in the literature about kisspeptin receptors and KiSS-1 levels of women who have undergone ovarian transplant. However, it has been determined that conditions causing loss of function of the kisspeptin or GPR54 receptor can cause hypogonadotropic hypogonadism, with loss of pubertal development and reproductive activity, a finding that suggests this system is necessary for the activity of gonadotropins. It is known that ovary-derived steroids play an important role in the release of gonadotropins. Accordingly, it is evident that ovarian functional disorders will cause changes in kisspeptin receptor and KiSS-1 levels. Increased expression of kisspeptin during luteinizing hormone elevation has been reported in steroids administered to rats after oophorectomy.²⁶ Navarro and colleagues²⁷ suggested that hypothalamic KiSS-1 expression was inhibited by estradiol treatment in oophorectomized rats. A number of previous studies have reported increases in estrogen receptor-alpha and KiSS-1 gene expression levels in arcuate kisspeptin neurons after oophorectomy.^28,29 We determined that kisspeptin receptor levels in ovarian tissue were lowest in the subcutaneous ovarian transplant group (628.57 ± 35.69 pg/mL; P < .05). Although there were numeric differences between peritoneum and subcutaneous groups with regard to kisspeptin receptor levels, these differences were not statistically significant. We observed no significant differences among experimental groups in terms of serum KiSS-1 concentrations.

Conclusions

Kisspeptin receptor levels were found to be lowest in the subcutaneous group. Although numeric dif­ferences between the peritoneum and subcutaneous groups were shown with regard to kisspeptin receptor levels, these were not statistically significant. Serum AMH levels were higher in the estrous group compared with the other groups. The experimental groups were comparable in terms of serum KiSS-1 concentrations.

In histopathologic tests, we observed no evidence of inflammation, fibrosis, or cysts in ovaries of the rat sham group. In the peritoneum and subcutaneous groups, pathologic conditions (inflammation, pig­mentation, follicular cysts, oil infiltration, surface inclusion cysts, and fibrosis) were observed. The subcutaneous group also displayed luteal cysts.

In histologic examinations of the physiological structures on the ovaries, statistically significant differences were found among the experimental groups in terms of other parameters, with the exception of atretic follicle counts. In the peritoneal and subcutaneous rat groups, no ovaries had secondary follicles. Tertiary follicle count and number of corpus luteum were also lowest in the peritoneal and subcutaneous groups.

With regard to estradiol concentrations, no signi­ficant differences were shown among the groups.

Immunohistochemical tests did not reveal KiSS-1-positive cell staining in granulosa cells of primordial, primary, secondary, and tertiary follicles of rats in all experimental groups. The most intense kisspeptin immunoreactivity was noted in early corpus luteum. The intensity of staining in the corpus luteum decreased in the peritoneal and subcutaneous groups compared with that shown in the sham and estrus groups, whereas the intensity of staining was increased in interstitial cells.

These data support the claim that serum AMH and kisspeptin levels are higher in rats in their estrus period and that concentrations of these hormones are low in heterotopic transplant of ovaries. Our data suggest that animals that received ovarian transplant subperitoneally have better results than those that received ovaries subcutaneously. In addition, we found that, although serum AMH levels were lower in the transplant groups versus the estrous group, it did not reach a level of zero. This result can be interpreted as the continuation of ovarian activities after transplant. With no publications on the measurement of AMH levels after ovarian transplant, we suggest that AMH levels should be evaluated in other animal species and especially in women who receive ovarian transplant.

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