INTRODUCTION

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide mediating the synthesis and release of pituitary luteinizing hormone (LH). Such polypeptides act mainly on the anterior pituitary (adenohypophysis), causing a rapid transitory increase in the release of LH, prolactin, somatotropin and follicle-stimulating hormone (FSH). By the late 1980s, superactive GnRH analogs and related depot medications have become widely available and were used for medical hypophysiectomy (suppression of LH and FSH secretion), which led to functional suppression of the male and female gonads. The strategy of reversible medical castration has been successfully implemented in treatment of various sex hormone-dependent diseases, including prostate cancer and premenopausal breast cancer, as well as endometriosis and uterine myoma. Estrogen withdrawal due to reversible medical castration by GnRH agonists has also been used as a conservative treatment for early endometrial cancer and its precancerous conditions in young women wishing to preserve their fertility [1].

However, when GnRH agonists are administered at a long-term basis and in supraphysiological doses, the paradoxical effect of pituitary hyperstimulation becomes evident. The metabolic mechanisms include pituitary desensitization to GnRH, suppression of GnRH receptors in target cells, a sharp decrease in the volume of LH secreted, disruption of physiological feedback mechanisms, with all these events being accompanied by a decrease in gonadotropin release. In growing female rats (aged 30–40 days), delayed vaginal opening and the inhibition of ovarian and uterine weight augmentation were detected when using GnRH analogs. These effects are used to develop new contraceptive methods, to treat endometriosis and precocious puberty, as well as in preparing for in vitro fertilization (IVF). GnRH agonists reliably induce anovulation and open the prospect of reversible pseudomenopause, which is essentially devoid of side effects. GnRH analogs have a considerable impact on the treatment of various estrogen-dependent gynecological diseases (Figs. 1, 2) [2–11].

In addition, GnRH analogs find use in the treatment of a wide range of sex hormone-related oncological processes, including advanced ovarian cancer (Fig. 1). Importantly, the effect of ovarian suppression in the treatment of oncological and gynecological diseases by GnRH agonists is reversible [2–6].

In children with precocious puberty, monthly intramuscular (i.m.) or subcutaneous (s.c.) injections of long-acting leuprorelin at doses from 3.75 to 15 mg reduce the mean growth rate and degree of manifestation of puberty signs, as well as increase predicted adult height vs. baseline measurements. Although the effect of medication on definitive adult height is predicted based on available data and requires confirmation in long-term follow-up studies, the lack of effective alternatives makes GnRH analogs the first-line therapy for children with such a rare disease [3].

The GnRH agonist MR-409, at a dose of 5 or 10 µg/mouse/day s.c., significantly reduces the lethality, severity of ischemic stroke, and degree of hippocampal atrophy, as well as improves neurofunctional recovery in animals with experimental transient middle cerebral artery occlusion. The drug can stimulate endogenous neurogenesis and improve neuroplasticity loss in experimental stroke. MR-409 also enhances proliferation and inhibits apoptosis of neural stem cells under conditions of restricted oxygen and glucose reperfusion [12]. Furthermore, GnRH agonists exert beneficial effects on animals with a model of ischemic and non-ischemic heart failure, including in chronic kidney disease [13].

Interestingly, as reported recently, both GnRH agonists and antagonists have a pronounced anti-inflammatory effect. The drugs from the group of antagonists suppress the formation of infiltrates in the lungs of rats with pneumonia. The results of ex vivo studies have shown that GnRH agonists and antagonists inhibit the lipopolysaccharide-induced production of pro-inflammatory and oxidative factors in isolated mouse colonic samples. In vivo, both groups of drugs are also able to reduce the response to nociceptive stimulation in the hot plate test. There has also been documented a decrease in the sensitivity to acute and sustained inflammatory stimulation in male mice in the formalin test and dextran sodium sulfate colitis model: the clinical symptoms, signs of histopathological injury and presence of pro-inflammatory and oxidative markers in colonic samples were clearly attenuated. Moreover, GnRH antagonists have more pronounced anti-inflammatory properties [14, 15].

Activation of the GnRH receptor, which has been found in retinal ganglion cells of adult rats, may relate to the preservation of these cells during inflammation. After optic nerve damage, s.c. injection of the GnRH agonist MR-409 or antagonist MIA-602 promoted retinal ganglion cell survival, which may be due to the additive effect on macrophage activation [16].

To summarize the results of studies on GnRH effects on the reproductive system of women and female animals, we carried out a literature search in PubMed and PubMed Health databases (www.ncbi.nlm.nih.gov). The data on clinical and experimental treatment of endometriosis, GnRH application for IVF and improving the chance of getting pregnant were excluded from the review. Also disregarded were the works on artificial insemination in veterinary medicine, fertility enhancement and ovulatory cycle synchronization.

Fig. 1.
figure 1

Main effects of GnRH analogs in clinical obstetrics and gynecology and in experiment.

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Fig. 2.
figure 2

The impact of GnRH analogs on the reproductive organs of women and female animals.

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EFFECT OF GONADOTROPIN-RELEASING HORMONE AGONISTS ON THE OVARIES IN WOMEN AND FEMALE EXPERIMENTAL ANIMALS

The use of the GnRH agonist buserelin to induce a state of reversible hypogonadotropic hypogonadism prior to the administration of exogenous gonadotropins is a promising strategy in the treatment of infertility associated with polycystic ovary syndrome and other conditions underlain by a dysfunction of these organs. This approach also clearly improves the effectiveness of IVF programs. The pilot studies suggest a potential utility of GnRH as a female contraceptive when used intermittently in combination with a progestagen. Buserelin represents the first-line treatment for precocious puberty [2].

A highly active GnRH analog NOE766 was administered to healthy women in a double-blind trial. According to the randomization plan, each woman received 10 µg NOE766 or placebo on day 1 to 14 of the menstrual cycle. Ovulation was suppressed for at least two weeks in all subjects who received the drug. An initially intense LH release, as measured 4 h after injection, decreased within 3 days by about 50% and remained at this level until the end of the experiment, while an initially high response to FSH was almost completely abolished during further treatment. In 3 of the 5 women who received the GnRH analog, the serum estradiol concentration was reduced significantly while in the in the remaining 2 subjects, it was reduced just slightly. Within 5 days after treatment cessation, the pituitary gland recovered its normal reactivity to GnRH. The authors concluded that follicle maturation was impaired due to NOE766-induced changes in physiological gonadotropin secretion [17]. Similar results were found when GnRH was administered to animals [15].

Kisspeptins are the most potent neurotransmitters that stimulate GnRH release. In small ruminants, kisspeptins or their analogs induced a surge of LH release followed by ovulation, while in horses, this transmitter only increased plasma LH levels but did not induce ovulation [19].

Cynomolgus monkeys Macaca fascicularis were divided into groups according to the method of ovarian stimulation (GnRH agonist or antagonist). In both groups, oocyte growth and maturation were induced by the administration of FSH and human chorionic gonadotropin. It was found that ovarian stimulation by a GnRH antagonist has some advantages for the given animal species over a controlled stimulation against the background of GnRH agonist [20].

Yano et al. [21] studied the direct effect of the GnRH agonist buserelin and GnRH antagonist cetrorelix on cell proliferation and differentiation in the rat ovarian follicles. Preovulatory follicles were obtained from immature rats and incubated in vitro in the presence or absence of human chorionic gonadotropin (hCG; 10 IU/mL), buserelin (10– 9 to 10–6 M), or cetrorelix (10–9 to 10–6 M) for 12 h. Buserelin induced meiotic oocyte maturation in a dose-dependent manner and also significantly stimulated the follicular production of prostaglandin E2 (PGE2) and progesterone, but not estradiol. Granulosa cells were obtained from the preovulatory follicles and cultured for 5 days. Both buserelin and cetrorelix inhibited the proliferation of such cells in a dose-dependent manner within the range of 10–10 to 10–5 M, with cetrorelix inducing the inhibition of mitotic activity stronger than buserelin. Electrophoretic analysis of genomic DNA isolated from granulosa cells treated with a GnRH analog at a concentration of 10–6 M revealed DNA fragments of an oligonucleosomal length, which are characteristic of apoptosis. The results demonstrate that both agonist and antagonist of GnRH inhibit granulosa cell proliferation via apoptosis, with the GnRH agonist additionally stimulating cell differentiation in the preovulatory follicle.

Ataya et al. [22] conducted two experiments on adult female rats to find out the effect of long-term application of GnRH agonists on the number and size distribution of ovarian follicles. Drug administration lasted for 52 days in the first experiment and 229 days in the second. Each 16th slice of the same ovary in each rat was examined under a light microscope coupled with a BioQuant computer image analysis system. After that, control and GnRH-treated rats were mated with the known male breeders and the offspring size and normality were evaluated. In the rats treated with a GnRH agonist, the total number of follicles, as well as the number and percentage of follicles with a diameter < 35 µm, were significantly higher, while the number and percentage of follicles with a diameter > 50 µm were significantly lower compared to the control group. The number of pregnant rats, as well as that of rat pups per litter, did not differ significantly in experimental and control animals. Thus, GnRH agonists inhibited the physiological process of follicle recruitment and loss, however, fertility persisted even after a long-term administration of GnRH agonists.

GnRH is expressed in the ovary and modulates the differentiation of its cell. Granulosa cells were obtained from immature rats and cultured in DMEM/F12 medium containing combinations of FSH, estradiol, and transforming growth factor-beta (TGF-beta) both in the presence and without GnRH. The GnRH analog leuprolide, administered at concentrations up to 5 × 10–11 M, caused a dose-dependent inhibition of 3H-thymidine inclusion in cells cultured in the presence of FSH (20 ng/mL) and TGF-beta (2.5 ng/mL). Similarly, a total inhibition of hormone-stimulated DNA synthesis was observed with another GnRH analog, buserelin, 1.58 ± 0.22 × 10–10 M) and native GnRH (1.4 ± 0.3 × 10–6 M). A competitive GnRH antagonist, antide, was used to neutralize the effects of GnRH agonists. At a dose of 10–8 M, antide is able to prevent DNA synthesis inhibition induced by 10–7 M leuprolide. In other words, GnRH can play a role in the regulation of rat granulosa cell proliferation during follicle development [23].

GnRH modulates differentiation of ovarian cells in different animal species. There was conducted a study of the direct effect of a GnRH agonist on the rate of apoptosis and steroidogenesis activity in porcine and human ovarian granulosa cells [24]. Cells were obtained from 6-month-old pigs, as well as from women after IVF, and cultured in a minimally required medium (MEM) added with 5% fetal bovine serum for 24 h. Buserelin was added to MEM at different concentrations (0.5; 50; 500 pg/mL and 5 ng/mL). Granulosa cell nuclei were examined by fluorescence microscopy after Hoechst 33258 staining. The GnRH directly increased apoptotic rate in granulosa-type cells. GnRH concentration in its clinical use turned out to be much higher than the concentration that induced apoptosis in cultured granulosa cells.

Takekida et al. [25] analyzed the effect of the GnRH agonist (10–9 M) on the proliferation, apoptosis and differentiation of cultured porcine granulosa cells obtained from the ovarian follicles at different stages of their development. The number of granulosa cells cultured in the presence of the GnRH agonist and being proliferating cell nuclear antigen (PCNA)-positive was lower compared to the cell cultures without the drug. However, the apoptotic rate was higher and the secretion of estradiol and progesterone by cultured cells was lower in the presence of the GnRH agonist. The inhibitory effect of the drug on cell proliferation was evident in cultured granulosa cells only from the small- and medium-sized, but not large, follicles. In contrast, the antiproliferative effect of the GnRH agonist on estradiol and progesterone secretion was only observed in cultured cells derived from the large follicles. However, the stimulating effect of the GnRH agonist on apoptosis was the same, no matter the stage of follicle development. These results demonstrate that the GnRH agonist has different effects on granulosa cells during follicle growth. The drug suppresses cell proliferation in the immature and steroidogenesis in the mature follicles; simultaneously, apoptosis of granulosa cells increases regardless of the stages of folliculogenesis.

Ovulation occurred in none of the perfused rabbit ovary preparations treated with buserelin or leuprolide acetate (102 to 104 ng/mL) in the absence of gonadotropin. GnRH agonists mediate meiosis resumption in follicular oocytes in a dose-dependent manner. In addition, the addition of GnRH to perfusate significantly increased the percentage of follicular oocytes showing the signs of degeneration compared to intact controls. The production of PGE2 and prostaglandin F2-alpha by perfused rabbit ovaries was significantly stimulated by GnRH administration, whereas no increase in progesterone or estradiol production was observed. GnRH acts directly in the rabbit ovary, inducing meiotic maturation in follicular oocytes, simultaneously enhancing their degeneration [26].

Preovulatory rabbit follicles were cultured in vitro with or without hCG (102 ng/mL), with buserelin (102 to 105 ng/mL) or leuprolide (102 to 105 ng/mL) for 14 h. GnRH agonists induced meiosis resumption in intrafollicular oocytes in a dose-dependent manner. The percentage of oocytes that achieved germinal vesicle breakdown after the treatment with 105 ng/mL buserelin (87.9 ± 6.3%) or 105 ng/mL leuprolide (86.0 ± 4.1%) was not significantly different from controls (87.3 ± 3.8%) after hCG administration. Mature oocytes were first detected within 2 h of GnRH agonist exposure. GnRH agonists significantly stimulated the production of both PGE2 and F2-alpha by preovulatory follicles (p < 0.01), although the levels of the secreted prostanoid did not differ significantly after exposure to different concentrations of GnRH agonists. Meiotic maturation of follicular oocytes after exposure to GnRH agonists began 2 h earlier than prostaglandin production and was dose-dependent, but oocyte degeneration increased at the same time. Prostaglandin production stimulated by GnRH agonists can be significantly reduced by indomethacin. However, oocyte maturity in the presence of a GnRH agonist together with indomethacin was not significantly different from that with the GnRH agonist alone. The simultaneous addition of 104 ng/mL GnRH antagonist blocked the stimulating effect of the GnRH agonist on oocyte maturation and prostaglandin production by mature follicles, and also reversed the rate of oocyte degeneration. The frequency of normal fertilization and early embryogenesis was significantly reduced in oocytes that matured in the presence of GnRH compared to those matured with hCG. Thus, GnRH agonist analogs induce meiosis resumption in follicle oocytes of the rabbit ovaries via a mechanism different from prostaglandin stimulation. The oocytes matured in vitro in the presence of GnRH are not necessarily cytoplasmically mature [27].

An implant with a retarded release of the GnRH agonist deslorelin (4.7 mg) was sewn subcutaneously to 13 rabbits to study its effect on ovarian function. Of them, 7 animals, before the onset of puberty, were introduced with a subcutaneous implant releasing GnRH over 273 days (group 1). After the resumption of ovarian function had been confirmed, the drug sources were reimplanted at the age of 430 days. Six adult rabbits (> 177 days; group 2) received deslorelin implants with a 273-day release. Ovarian function before, during, and after implant treatment was assessed by measuring serum progesterone levels in peripheral blood 10 days after the control injection of short-acting GnRH (0.8 µg buserelin i.m.). In group 1, the animals were subjected to ovariohysterectomy during the second course of drug administration, and the ovaries were then examined histologically. The ovaries were found to contain non-atretic and atretic follicles at different developmental stages but lacking an active yellow body. Group 2 animals underwent ovariohysterectomy 2 to 12 months after implant removal. Their ovaries contained follicles at different stages of development, as well as the white bodies. Consequently, a reversible suppression of ovarian functions can be achieved in female rabbits by using implants with a retarded GnRH release, introduced before or after the onset of puberty [28].

Thus, GnRH agonists suppress ovulation and induce a menopause-like conditions in women and female animals when used at a long-term basis. Infusion of GnRH agonists is a simple method to suppress LH and FSH production, as well as follicle development. In the ovaries, granulosa cells are inhibited via suppression of DNA synthesis and induction of apoptosis with simultaneous stimulation of cell differentiation in preovulatory follicles (Fig. 1). This condition is reversible, fertility is preserved, and after treatment cessation, the pituitary gland restores its ability to respond to drug releasing in less than no time.

AGONISTS OF GONADOTROPIN-RELEASING HORMONE AND THE UTERUS IN CLINICAL AND EXPERIMENTAL MEDICINE

As mentioned above, GnRH analogs are used to treat a wide spectrum of sex hormone-related diseases, including endometriosis and precocious puberty [3]. However, when used at a long-term basis, they can cause pituitary desensitization and/or downregulation, leading to a suppression of circulating gonadotropin and sex hormone levels.

In patients with uterine myoma, leuprorelin or goserelin administration reduces uterine volume and disease-associated symptoms, but, as with other GnRH analogs, these effects disappear after treatment discontinuation. Due to the therapy, all patients achieved a menopause-like condition, as evidenced by hot flashes, depression, vaginal dryness, hysteroscopic signs of endometrial atrophy, and appropriate changes in the hormonal parameters (FSH, LH, estradiol levels). The drugs based on GnRH analogs can be useful for reducing the volume of surgical intervention due to a reduction in the size of uterine myoma, although this does not completely exclude the surgery as such [2, 3]. Leuprorelin is an effective alternative to other treatments for women with endometriosis, although the recommended duration of its use in this clinical situation is limited to 6 months because it reduces bone mineral density [3].

Female Wistar rats in estrus or diestrus (as determined by vaginal cytology) were injected daily with 20 mg of buserelin acetate s.c. for 4, 8, or 12 days. The rats were withdrawn from the experiment 24 h or 5 days after the end of the treatment course. The control group of animals received a drug vehicle for 12 days. A progressive hypotrophy of uterine tissues arose during observation and was accompanied by estrogenic hyperactivity for 5 days after the end of the experiment. Vaginal cytology and endometrial histology revealed a heavily vacuolized mucosa and glandular epithelium; the cyst margins and endometrial stroma were densely infiltrated with eosinophils. Buserelin appears to cause a progressive blockade of gonadotropin secretion and an important recoil effect, with an enhanced estrogen release manifesting itself as soon as the first estrous cycle after the end of drug administration [29].

In addition to intrauterine antibiotic therapy, 30 cows with postpartum endometritis were injected with 20 µg of the GnRH analog buserelin between postpartum days 10 and 12, and then, 10 days later, with 500 µg of the prostaglandin F2-alpha analogue cloprostenol. Forty control animals were treated with in utero injections of antibiotics only. The uterine involution improved after hormonal treatment: on day 42 postpartum, it was complete in 93.3% of the animals treated with hormones and in 82.5% of the cows that received antibiotics only (p ≤ 0.05). Clinical recovery was 96.6% in the experimental group and 82.5% in the control group (p ≤ 0.05). The first pregnancy indices were significantly better in the animals treated hormonally than in the control animals (51.7 vs. 36.4%, p ≤ 0.05). However, the values of the general pregnancy indices and insemination index were not significantly improved after GnRH and prostaglandin F2-alpha administration. It was concluded that the consistent application of hormonal drugs in cows with postpartum endometritis positively influenced ovarian function and uterine involution, which led to the improvement of fertility indices [30].

Eight macaques were injected daily with 5 or 20 µg of a GnRH agonist for about one year to prevent ovulation. On the last day of treatment (7 animals) or during the mid luteal phase of the first menstrual cycle after treatment (1 animal), they were laparotomized. The uterine size was smaller (p < 0.01) compared to controls with a normal cycle (n = 6). A wedge-shaped fragment of the full-thickness anterior uterine wall was resected and examined histologically. In 5 agonist-treated monkeys, the endometrium matched an atrophic or resting proliferative type, but in 2 animals, it ranged from an early proliferative to a marked secretory type. All changes proved to be only benign. In a single animal studied in the luteal phase after treatment cessation, a normal secretory endometrium was found. The ovaries from 2 animals after GnRH agonist exposure were also examined histologically and found to consist of the follicles at different stages of maturation. The presence of endometrial activity in 2 of 8 GnRH agonist-treated monkeys accentuates the need to thoroughly assess the endometrium during therapy that includes repeated GnRH agonist administration in female patients [31].

The effects of GnRH antagonists, used in IVF protocols, on endometrial tissue remodeling, embryo implantation and early pregnancy programming are still poorly studied, arousing some concerns about the course of pregnancy and IVF outcomes for babies after GnRH antagonist therapy [32]. Recently, GnRH and a splice variant of the GnRH receptor 1 (GnRHR1) have been found to be expressed in human decidual stromal cells isolated from the decidual tissues of women at early terms of pregnancy, who have undergone surgical abortion [33]. It has been shown that GnRH agonist promotes the motility of human decidual stromal cells via the GnRH receptor and phosphorylation of the extracellular signal-regulated protein kinase 1/2, as well as JNK-dependent MMP-2 and MMP activation, which exerts a strong effect on embryo implantation [32].

Due to prolonged GnRH administration (up to 2 years with several cycles), endometrial hyperplasia and endometritis were observed in 5 of 7 rabbits. A histopathological examination of 4 animals castrated during induced pseudopregnancy revealed no signs of uterine alterations. Two ovariohysterectomized animals were diagnosed with endometritis 12 months after implant removal. Therefore, the development of age-related uterine pathology cannot be prevented by suppressing ovarian functions through the long-term use of GnRH drugs [28].

GnRH is expressed in human decidual stromal cells isolated from the decidual tissues of pregnant women who had undergone surgical abortion at early terms of pregnancy. The treatment of stromal cell elements with the GnRH antagonist JMR-132 induced apoptosis with increased caspase-3 and -9 activities and reduced cell viability in a time- and dose-dependent manner [32].

Rapid involution of the uterus after using GnRH analogs is able to delay or even arrest myoma progression with the disappearance of its symptoms, with uterine blood flow remaining unaffected. Against the background of uterine hypotrophy, the endometrium corresponds to an atrophic or resting proliferative type (Fig. 1). At the same time, there is also evidence to support quite opposite views, namely that long-term GnRH administration promotes endometrial hyperplasia and endometritis, and that the suppression of ovarian functions is unable to prevent the development of age-related uterine pathology. In any case, GnRH treatment requires a close monitoring of the endometrium. It is also possible to administer GnRH agonists to accelerate postpartum uterine recovery, even during antibiotic therapy for endometritis.

ANTITUMOR EFFECT OF GONADOTROPIN-RELEASING HORMONE AGONISTS

GnRH receptors are expressed in about 80% of human endometrial and ovarian cancers and account for more than 50% of breast cancers, including triple-negative breast cancer (34). Except for the pituitary and reproductive organs, no other organs or hematopoietic stem cells express GnRHR. Thus, these receptors can be considered as an ideal target for a personalized medical approach in cancer therapy.

GnRH analogs act on hormone-dependent tumors and influence their growth. Some long-acting drugs have porved effective in the treatment of ovarian tumors. Modern GnRH analogs demonstrate very low endocrine but high antitumor effects both in in vitro and in vivo experiments. Tritium-labeled GnRH derivatives reveal specific binding sites in human tumor cell lines. Third-generation GnRH analogs with an effective selective antitumor activity have been synthesized that do not alter the rat ovarian cycle but inhibit colony formation in cancer cell lines and have a considerable antiproliferative effect (Fig. 1). Using radioactively labeled analogs of peptide hormones, it has been found that human tumor cell lines and xenografts specifically bind GnRH conjugates. New GnRH analogs, acting without any hormonal effect, may represent a breakthrough in the study of antitumor peptides having a direct effect on tumor cells [35].

It is necessary to particularly highlight the literature data on successful treatment of uterine cancer metastases to the lungs, bones and other organs with GnRH agonists [36, 37, 38].

The number of human ovarian cancer cells (HRA line) and their 3H-thymidine uptake significantly increased after FSH addition, although the effect was suppressed by buserelin. FSH and GnRH receptors were identified in HRA cells, and their number was significantly reduced by buserelin treatment [39]. The proliferation of HHUA (endometrial carcinoma) cells has been studied in low- and high-density cultures under conditions of buserelin application. The drug negatively affected colony formation in a dose-dependent manner at low cell densities, but was ineffective at high cell densities. HHUA cell culture medium inhibited the effect of buserelin. These results suggest that cell lines secrete some substances during culturing, which regulate cell proliferation, and that these substances can also modify the effects of this GnRH analog [40].

An ovary implanted to the spleen of an ovariectomized rat develops a luteinized tumor that grows in response to gonadotropins. Buserelin restrains tumor growth, probably due to a direct inhibitory effect on luteoma cells. Cells derived from an experimental luteinized ovarian tumor are more sensitive to the endocrine effect of GnRH compared to intact luteal cells. The transformation of ovarian cells into luteoma implies the acquisition of new characteristics in the system of GnRH receptor generation [41].

In vitro, buserelin induced a decrease in the number of ovarian cells obtained from prepubertal rats, as well as cells obtained from the ovarian lutein cell tumor, to the same extent. Although basal levels of apoptosis were higher in ovarian cells than in lutein cell tumor cells, buserelin-induced apoptosis was only detected in luteoma cells after a 48-h exposure [42].

GnRH analogs can exert direct antitumor effects on cells of various ovarian cancer cell lines. Buserelin caused a statistically significant reduction in cell growth in two of the six cell lines of this cancer, although no dose dependence was observed. Leuprolide (a GnRH agonist) led to a significant dose-dependent growth inhibition in all six cell lines when doses were increased up to supraphysiological levels, but showed no significant inhibition at clinically allowable doses. At the same doses, antide (a GnRH antagonist) had no effect on tumor growth. A competitive interaction analysis revealed no specific binding of the drugs under study to any of the six ovarian cancer cell lines tested [43].

Slotman et al. [44] only observed a small direct inhibitory effect of buserelin at high concentrations on the proliferation of three human ovarian cancer cell lines. The authors concluded that a direct antitumor effect is unlikely to be the main mechanism of GnRH action in cancer treatment.

Meanwhile, there is evidence that buserelin does not inhibit the growth of DMBA-induced rat ovarian adenocarcinoma, although an enhanced necrosis of the tumor core, a decrease in the number of tumor cells, and connective tissue proliferation were observed histologically in the buserelin-treated rat group. A daily in vivo buserelin administration significantly suppressed the release of FSH, LH, and progesterone compared to the control group. In vitro, buserelin suppressed FSH-induced proliferation of DMBA cells [45].

Eighteen cases of malignant ovarian neoplasms were studied experimentally to find out the possible role of gonadotropins in tumor development. Twelve cases of serous cystadenocarcinoma, 2 of mucinous cystadenocarcinoma, 2 of endometrioid carcinoma, one of Brenner tumor, and one of yolk sac tumor were examined after having been implanted under the kidney capsule of female mice to determine their response to buserelin. The results showed that the size of the xenografts increased (p < 0.05) in the group treated with the GnRH analog. Apparently, gonadotropins play a certain role in the oncogenesis stimulation of ovarian malignancies by binding to specific receptors [46].

Since GnRH agonists inhibit ovarian function and induce uterine involution, these drugs can be successfully used to treat benign and malignant processes in these organs, as well as to affect metastases (Fig. 1). However, at the same time, there are studies showing that a direct effect of GnRH on tumors is poorly pronounced. There is also an opinion that gonadotropins can even stimulate the development of ovarian cancer by affecting certain receptors.

EFFECTS OF GONADOTROPI-RELEASING HORMONE DRUGS ON THE REPRODUCTIVE ORGANS DURING CHEMOTHERAPY

To explore the mechanism of the protective effect of GnRH agonists against chemotherapy-induced ovarian damage, female rats were implanted with 1 mg of the GnRH agonist (zoladex pellets). All rats were additionally implanted with 3H-thymidine-loaded osmotic minipumps or slow-release pellets 48 h before the withdrawal from the experiment. Five days after pellet implantation, GnRH significantly reduced 3H-thymidine uptake by the ovaries. Autoradiography showed that almost all 3H-thymidine was localized to granulosa cells. It cannot be ruled out that GnRH suppresses the mitotic activity of ovarian cells. Since cytotoxic agents affect preferentially rapidly dividing cells, these results may represent a mechanism of ovarian protection during chemotherapy (Fig. 1) [47].

The possibility of reducing the ovariotoxicity of the antitumor drug etoposide by buserelin was investigated in female Wistar rats. A quantitative analysis of the structural and functional elements of the ovaries on whole-organ serial sections showed that 3 months after a combined etoposide/buserelin treatment, the morphological picture of the ovarian glands was indistinguishable from that in intact animals of the same age, whereas etoposide monotherapy resulted in an earlier development of atrophic processes. Six months after treatment, the number of bi- and multilayer follicles was significantly higher in the rats received the combined therapy compared to those treated with etoposide alone [48].

A meta-analysis of 13 randomized controlled trials on a comparative assessment of the effects of co-application of GnRH analogs and chemotherapy (609 testees) with those of chemotherapy alone (599 testees) has provided evidence (albeit of low quality, as rated by the authors themselves) that GnRH agonists are advisable to be used before and/or during chemotherapy to reduce the risk of primary ovarian failure and increase the chance of spontaneous pregnancy in the short run [49].

However, in a study by Waxman et al. [50], buserelin proved ineffective in preserving fertility during cytotoxic chemotherapy. Eighteen women were randomly allocated to receive a GnRH agonist before and during cytotoxic chemotherapy for advanced Hodgkin’s disease. Buserelin was prescribed to 8 women as a single injection regimen. A standard test for GnRH was performed a week before and on day 1 of each cycle of chemotherapy. The regimen used resulted in an initial suppression of FSH peak responses to GnRH, which was not sustained throughout the follow-up. During the subsequent 3-year follow-up evaluation after buserelin treatment completion, amenorrhea was reported in 4 of 8 female patients and in 6 of 9 control women.

Ineffectiveness of GnRH analogs for ovarian protection during chemotherapy was confirmed by Elgindy et al. [51] in a review of an ample data with a meta-analysis. The authors compared the published results of randomized controlled trials of ovarian functional recovery between groups of female patients after chemotherapy with and without GnRH agonists. It was concluded that the inclusion of GnRH analogs into the treatment protocol is unreliable in terms of fertility preservation.

It is also necessary to mention the works showing that available evidence is insufficient to conclude on the effectiveness or otherwise of ovarian protection through co-application of GnRH agonists and chemotherapeutic drugs. The authors emphasize the need for further studies, which, among other things, should take into account pregnancy rates and changes in the effectiveness of antitumor therapy (52).

Thus, there is a certain inconsistency when analyzing the results of using GnRH analogs as protectors against damaging effects of chemotherapy on the human reproductive system. The idea of such use is again related to the suppression of ovarian and endometrial cell activity, whereas the damaging factors have a stronger adverse effect exactly on the proliferatively and functionally active cell elements. Most scientists report good protective potential of GnRH agonists during chemotherapy, however, there are also data on poor or no protective efficacy of the drugs of this group.

CONCLUSIONS

It can be concluded that the main studies of the effects of GnRH agonists on the reproductive organs of women and female experimental animals were carried out yet at the end of the past century. Current studies are mainly focused on specific features of using GnRH analogs and the results of applying new drugs for the treatment of certain pathologies. Many authors point out that GnRH agonists, when used for a long time, suppress ovulation and cause a menopause-like condition. Infusion of GnRH agonists is a simple method for suppressing LH and FSH production and follicle development. In the ovaries, there is an inhibition of granulosa cells through suppression of DNA synthesis and induction of apoptosis paralleled by the stimulation of cell differentiation in preovulatory follicles. This condition is reversible and fertility is preserved: after treatment completion, the pituitary gland restores very quickly its ability to respond to GnRH release. Due to the rapid uterine involution after the use of GnRH analogs, a delay or even arrest of myoma progression with the disappearance of its symptoms is possible, with uterine blood flow remaining unchanged. Against the background of uterine hypotrophy, the endometrium corresponds to an atrophic or resting proliferative type. At the same time, there is quite opposite evidence that long-term GnRH application contributes to endometrial hyperplasia and endometritis, and that the suppression of ovarian functions does not prevent the development of age-related uterine pathologies. In any case, GnRH treatment requires close monitoring of the endometrium. It is also possible to administer GnRH agonists to accelerate postpartum uterine recovery even with a simultaneous antibiotic therapy for endometritis. GnRH drugs are finding successful use in treating benign and malignant processes in the ovaries and uterus, as well as to affect metastases. At the same time, there are studies showing that there is little direct effect of GnRH on tumors. There is an opinion that gonadotropins may even stimulate ovarian neoplastic transformation by acting on certain receptors. Most authors report a good protective ability of GnRH agonists during chemotherapy, but there is conflicting evidence of poor or no protective efficacy of this group of drugs. Anyway, the inconsistency of publications on each of the aspects of GnRH effects indicates that they are still awaiting not only applied, but also fundamental investigation.