The use of GnRH analogs in preserving ovarian function during chemotherapy

The literature has always been controversial on the use of gonadotropin-releasing hormone agonists in preserving fertility in women of childbearing age after chemotherapy; thereby, in this article, we will be discussing its use in preserving fertility. When it comes to preserving fertility, it is crucial to consider all available options in this topic due to its very sensitive nature, thereby we have found that while a lot of trials favor the use of gonadotropin-releasing hormone agonists, the lack of proper follow-up and long-term trials renders its use highly debatable, and since the longest follow-up trial showed non-significant results, it also opens the floor for debate on whether this short-term benefit is worth adding another drug to the regimen or not. As described in this review, while the use of gonadotropin-releasing hormone agonists is beneficial in a lot of studies, the lack of long-term reports still makes its use debatable, thereby more trials should be done.


Background
The management of malignancy in women in their reproductive years may necessitate a surgical, radiological, or a cytotoxic (chemotherapy) approach which exposes the female to dangerous and even toxic doses that will ultimately result in gonadal damage and dysfunction.
Prior to initiating potentially gonadotoxic therapy, physicians should discuss the risk of treatment-induced infertility and possible interventions to preserve fertility [1]. This discussion should occur soon after diagnosis since some interventions to preserve fertility take time and could delay the start of treatment. The ASCO's (American Society of Clinical Oncology) committee opinion stated in 2006 and reaffirmed in 2013 that as part of education and informed consent before cancer therapy, healthcare providers (including medical oncologists, radiation oncologists, gynecologic oncologists, urologists, hematologists, pediatric oncologists, and surgeons) should address the possibility of infertility with patients treated during their reproductive years (or with parents or guardians of children) and be prepared to discuss fertility preservation options and/or to refer all potential patients to appropriate reproductive specialists. The ASCO's committee also stated in 2018 that the guidelines emphasize the use of measures to preserve fertility in young women with cancer, and such measures include assisted reproductive techniques and ovarian protection through the use of GnRH agonists. Although patients may be focused initially on their cancer diagnosis, it is encouraged to advise patients regarding potential threats to fertility as early as possible in the treatment process so as to allow for the widest array of options for fertility preservation [2].
This emphasizes the importance of a multidisciplinary approach and collaborative work between oncologists and reproductive medicine specialists in tackling such a crucial and sensitive issue.
Page 2 of 11 Odeh et al. Middle East Fertil Soc J (2021) 26:42 In general, the ovaries of women who receive chemotherapy have a decreased number of primordial follicles which will result even in a greater decrease in the numbers of larger maturing follicles indicating a greater chemotherapeutic effect on follicular development rather than on the oocyte itself. Many young women develop amenorrhea during chemotherapy, and this is what is called chemotherapy-induced amenorrhea which on its own has a significant independent clinical impact (type and dose-dependent) on survival [3]. Reviewing the results of the International Breast Cancer Study Group (IBCSG) Trial VI showed that adjuvant chemotherapyinduced amenorrhea has been shown to be associated with reduced relapses and improved disease-free and overall survival for premenopausal breast cancer patients and is especially significant for node-positive breast cancer [4].
In general, women younger than age 40 years are more likely to retain their menstrual cycles than those older than 40 as they have a larger pool of follicles [5].
Multiple approaches are available for fertility preservation in women undergoing gonadotoxic treatment, these include the following: 1-Cryopreservation of embryos 2-Cryopreservation of mature and immature oocytes 3-Cryopreservation of ovarian tissue 4-Ovarian transposition 5-Gonadal shielding during radiation therapy 6-The use of GnRH analogs in patients with chemotherapy The efficacy of the use of the GnRH analogs for preserving ovarian function during chemotherapy was always debated in the literature with different findings across multiple randomized controlled trials, with some data regarded as heterogeneous due to the fact that there was no uniform or an absolute definition for premature ovarian failure with many studies referring to amenorrhea as a reflector of ovarian function as well as the scarcity of data on future patient fertility, pregnancy, as well as pregnancy outcomes.
In this review, we will be tackling the issue of the use of the GnRH analogs in preserving ovarian function in patients receiving chemotherapy.

Biology of GnRH analogs
In 1977, the structure of the GnRH was properly identified by Drs. Guillemin and Schally [6]. A decapeptide (pyro Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 ) synthesized from a 92-amino acid preprohormone in the preoptic nucleus in the hypothalamus. The portal blood carries the GnRH to the pitui tary gland, which contains the gonad otrope cells, where GnRH activates its own recep tor, gonad otrop in-relea sing hormo ne recep tor (GnRHR), to release FSH and LH.
The half-life of GnRH is 2-4 min as it is degraded by peptidase and cleared by glomerular filtration [7], and this was probably the main reason for developing longeracting agonistic derivatives to increase their duration of action [8]. The GnRH analog causes an initial flare effect that increases gonadotropin levels and causes ovarian stimulation followed by a hypogonadotrophic state within 1-3 weeks of administration [7].
The administration of LHRHa is associated with some adverse effects (e.g., headache, hot flashes, vaginal dryness, sweating, mood changes, insomnia, urogenital symptoms, and thromboembolic events).
Safety concerns regarding its use were raised suggesting a possible increase in the side effects during cytotoxic therapy, a possible detrimental effect of the lack of treatment-induced amenorrhea on prognosis, and a potential negative interaction with chemotherapy.
However, the three available randomized studies [9][10][11] that investigated the impact of adding concurrent ovarian function suppression to chemotherapy did not demonstrate any difference in patients' prognosis.

Mechanism of action in fertility preservation
In the adult ovary, > 90% of the ovarian reserve is made up of primordial follicles in the resting stage prophase I. Growth initiation of follicles is initially an FSH-independent process and gonadotropin-dependent growth would not occur until later on (antral phase) [12]. Because profound ovarian suppression may take several weeks to achieve, it is unlikely that sufficient lowering of gonadotropins will be achieved within the short time available before the initiation of chemotherapy. Also, if GnRH analogs are given during the follicular phase of the cycle, they may actually cause a flare effect and create the opposite of the desired impact and defeat the purpose of actually suppressing ovarian function during chemotherapy treatment.
This raised many questions regarding how GnRH analogs actually work in fertility preservation.
A proposed intervention in mice showed that gonadotropins enhance caspase-3 and caspase-7 and apoptosis in the theca-interstitial cells of rat preovulatory follicles in culture. The elevations in caspase-3 and caspase-7 activities in theca-interstitial cells were accompanied by an increase in apoptosis [13], since theca-interstitial cells are important for follicle development, this increase in apoptosis might explain the preservation of preovulatory follicles, as this can help them maintain a relatively dormant state. Thus, pituitary desensitization, induced by GnRH agonist administration, prevents the secretion of growth factors by the FSH-dependent follicles, thus secondarily preserving more primordial follicles in the "dormant" stage and minimizing their unidirectional maturation and ultimate destruction by alkylating agents Two recent publications found that the proliferation of primordial germ cells is gonadotropin-dependent and is mediated by Akt/phosphatase and tensin homolog deleted on chromosome ten (PTEN) signaling. High concentrations of estrogens stimulate mouse primordial germ cell growth in vitro. Moreover, estrogens stimulate the transcription of the "Steel" gene and the production of c-Kit ligand in gonadal somatic cells, and this growth factor is likely to be responsible for the observed stimulation of primordial germ cell growth via an Akt/PTEN pathway [14,15]. Although the initiation of primordial follicle growth and the early stages of folliculogenesis can occur without gonadotropins, FSH may affect the rate of preantral follicle growth as well; therefore, the assumption that early follicles are gonadotropin independent may need re-evaluation [15][16][17][18].
Another proposed modality of action aims to decrease the utero ovarian perfusion created by high estrogen states in the body. High estrogen concentrations significantly increased ovarian perfusion and vessel endothelial area in a rat model of ovarian hyperstimulation, and this effect was significantly and dosedependently inhibited by administration of a GnRH agonist.
Moreover, it has been shown that human gonads contain independent GnRH receptors. GnRH-I and GnRH-II receptor activation may result in decreased apoptosis. Whether the GnRH agonist effect is direct on the oocyte-cumulus complex or on the granulosa cells themselves, this topic in particular requires further assessment [19].
Another possibility is that GnRH agonists may upregulate an intragonadal antiapoptotic molecule such as sphingosine-1-phosphate (S1P). Tilly et al. [20] have identified several molecules that are required for chemotherapy-induced oocyte apoptosis. While much of their work has relied on mice genes, they have identified a lipid antagonist of the proapoptotic second messenger ceramide, S1P, as a protective molecule [21].

Clinical data
In this review, we will be discussing the use of GnRH analogs in patients with breast cancer and hematological malignancies, since breast cancer is the most common cancer in women, and due to lack of available data, only hematological malignancies were discussed.

Breast cancer
Del Maestro et al. [22] demonstrated that the use of triptorelin-induced temporary ovarian suppression during chemotherapy in premenopausal patients with early-stage breast cancer reduced the occurrence of chemotherapyinduced early menopause. The study group looked at the incidence of early menopause in young (aged 18-45) patients with breast cancer undergoing adjuvant or neoadjuvant chemotherapy in a randomized controlled trial in 16 sites in Italy where patients were randomized into receiving chemotherapy alone vs chemotherapy and triptorelin administered intramuscularly at a dose of 3.75 mg at least 1 week before the start of chemotherapy and then every 4 weeks for the duration of chemotherapy. Early menopause was defined as no resumption of menstrual activity and postmenopausal levels of FSH and estradiol 1 year after the last cycle of chemotherapy. The study included hormone receptor-positive as well as hormone receptor-negative breast cancer patients where hormone receptor-positive patients received adjuvant treatment with tamoxifen for up to 5 years. There was no significant difference between the chemotherapy regimens their patients received, either CMF-based (cyclophosphamide, methotrexate, and fluorouracil), anthracycline-based, or anthracycline-taxane-based treatment. With 133 patients randomized to chemotherapy alone and 148 patients randomized to chemotherapy plus triptorelin, the rate of early menopause was 25.9% in the chemotherapy-alone group and 8.9% in the chemotherapy plus triptorelin group (P = 0.001) thus an attributable risk reduction of 17% (95% confidence interval, − 26% to − 7.9%; P < .001). In a multivariate analysis, only the treatment with triptorelin was associated with a significant reduction of the risk of developing early menopause with an odds ratio for treatment-related early menopause of 0.28 (95% confidence interval, 0.14 to 0.59; P < .001).
Moore et al. [23] demonstrated in a phase 3 trial that ovarian failure rate was significantly less in patients receiving goserelin starting 1 week before the start of the chemotherapy regimen than those in the control group (odds ratio, 0.30; 95% confidence interval [CI], 0.09 to 0.97; P = 0.04). Furthermore, after a 5-year follow-up, they also showed that those who received goserelin were more likely to get pregnant than the control group (odds ratio, 2.45; 95% CI, 1.09 to 5.51; P = 0.03), without the presence of a difference in pregnancy complications.
In 2012, Munster et al. [24] looked at premenopausal women aged 44 years or younger which were randomly assigned to receive either triptorelin or no triptorelin during neoadjuvant chemotherapy and assessed the preservation of their ovarian function through and after receiving chemotherapy. These patients were stratified by age (< 35, 35 to 39, > 39 years), estrogen receptor status, In a randomized, parallel-group study, the Anglo Celtic Group OPTION trial by Leonard et al. [25] which recruited 227 patients, goserelin reduced the prevalence of amenorrhoea between 12 and 24 months to 22% vs 38% in the control group (P = 0.015) and the prevalence of POI to 18.5% vs 34.8% in the control group (P = 0.048). Follicle-stimulating hormone concentrations were also lower in all women treated with goserelin at both 12 and 24 months (P = 0.027, P = 0.001, respectively). However, the use in women above the age of 40 proved to be nonsignificant, further strengthening the evidence supporting the use of GnRHa, but that still does not deny the fact that longer-term outcomes are yet to be studied and that the efficacy in preserving ovarian function is still limited.
A meta-analysis on 14 RCTs [26] showed that the use of GnRHa is favorable as it significantly reduced the risk of POF when given during chemotherapy (OR 0.36, 95% CI 0.23-0.57, P < 0.001). Only 5 studies out of the 14 RCTs studied post-chemotherapy pregnancies, which was also deemed significant (OR 1.83, 95% CI 1.02-3.28, P = 0.041). This calls for more trials to study the pregnancy rates after chemotherapy, as the current literature does not have much information on this specific variable.
Another meta-analysis done by Lambertini et al. [27] on 5 trials supported the use of GnRHa, but an interesting fact is that they also stated that a younger age (< 40 years) at diagnosis was a significant variable (adjusted OR, 0.35; 95% CI, 0.24-0.52; P < 0.001) when it came to the reduction of risk of developing chemotherapyinduced premature ovarian insufficiency (POI), thereby concluding that age is a major variable when it comes to the success of the use of GnRHa in the prevention of chemotherapy-induced POI.
One of the trials supporting the use of triptorelin, the PROMISE trial [28], specifically when it comes to the long term, which is one of the points that makes the use of GnRH agonists debatable, but it is worth mentioning that they also concluded that it does not affect pregnancy rates. The variable found significant was menstrual resumption at 5 years between the GnRHa and control group, 1.28 (95% CI, 0.98-1.68; P = 0.07) ( Table 1).

Hematological malignancies
Salama et al. [29] in a review addressed preserving fertility in female patients with hematological malignancies, in which they have discussed the use of GnRH agonist along with a lot of other methods. Given that they have stated that the use of GnRH agonists is debatable, it is worth noting that they have stated that it is not suitable for use in prepubertal females due to inactive HPO axis, does not require a delay in cancer treatment, should be carried out before and during chemotherapy, and that it does not protect against the gonadotoxic effects of radiotherapy. However, it was listed as debatable due to contradictory results.
In another retrospective cohort study, triptorelin (Decapeptyl) was administered monthly to 61 women with Hodgkin lymphoma who were under treatment from 1994 to 2006. And out of these 61 women, 50 recovered regular menses, and after the completion of treatment, 13 patients conceived successfully. Falorio et al. [30] concluded that the use of GnRHa may be useful in preventing ovarian damage and infertility in young women receiving polychemotherapy alone or in combination with subdiaphragmatic radiotherapy, but it is not effective in refractory or relapsed patients, since they have found a clear correlation between age at the time of treatment, advanced disease, cumulative therapeutic load, and ovarian failure.
Another study including data from more than 20 years ago was carried out by Driul et al. [31], where they aimed to assess ovarian function in those receiving GnRHa compared to those who did not in a case-control study in survivors of hematological malignancies, where they analyzed 124 patients between 1998 and 2007. The results showed that in those treated with GnRHa, 33% had post-treatment amenorrhea and 6% post-treatment pregnancies, compared to 49% and 4%, respectively, in the control group; however, they have In a multivariate analysis, only the treatment with triptorelin was associated with a significant reduction of the risk of developing early menopause with an odds ratio for treatment-related early menopause of 0.28 (95% confidence interval, 0.14 to 0.59; P < .001). Lack of follow-up to assess the end points.
Moore et al. [ Individual patient-level data from only five major randomized trials were included.  26:42 concluded that there was no statistical significance to emphasize on the use of GnRHa and that more evidence should be obtained. A retrospective cohort study compared 286 patients who received GnRH agonist with chemotherapy and 188 patients who were treated with chemotherapy alone, and in this study, the primary outcome was spontaneous pregnancies. The secondary outcome was cyclic ovarian function (COF) vs premature ovarian failure (POF). These outcomes were assessed 2 years or more after chemotherapy. Blumenfeld et al. [32] concluded that spontaneous conception is significantly achieved in the intervention group (P = .0004, OR 3.12, 95% CI 1.7-5.8), and adding to that preservation COF (P = .0001), thereby adding to the literature in supporting the use of GnRHa. Furthermore, 123 healthy newborns were born in the intervention group, compared to 40 in the control group.
In a prospective non-randomized case-control study, Blumenfeld et al. [33] elaborated on the effect of co-treatment with GnRHa on ovarian damage and concluded that it may reduce ovarian damage significantly. This study recruited 115 female patients with Hodgkin lymphoma, where 65 patients received a monthly injection of GnRHa throughout and before starting chemotherapy, up to a maximum of 6 months. The results showed that the resumption of regular menses was significantly seen in the intervention group; however, no significance was seen on the preservation of COF, which contradicts previous studies, and these results further emphasize the need for more trials on the use of GnRHa.
Taking it a step further, in a study aiming to compare the rate of POF after stem cell transplantation (SCT) in women receiving GnRHa in conjunction with gonadotoxic chemotherapy, Blumenfeld et al. [34] showed that in 83 patients, 18 out of the 47 patients receiving GnRHa resumed cyclic ovarian function, compared with 4 out of the 36 not receiving GnRHa. There were no significant differences in age, chemotherapy treatment, or diagnoses between the study and control groups. They concluded that GnRHa cotreatment in conjunction with conditioning chemotherapy before SCT may significantly decrease the gonadotoxicity and POF from 82 to 33% in lymphoma but not in leukemia patients.
On the other hand, Demeestere et al. [35] showed that even though on the short term, the ovarian function might be better in a group receiving GnRHa when compared to a control group (3.14 ± 0.80 ng/mL at inclusion vs 1.26 ± 0.3 ng/mL after 2 to 4 years, P = .039); this significance was no longer seen when it came to a longterm follow-up (1.58 ± 0.38 ng/mL, P = .520). In their study, they signified the fact that more trials should be done to assess the long-term effects of the use of GnRHa in preserving fertility, and this calls for the discussion of whether this short-term improvement is worth the addition of GnRHa or not (Table 2).

Conclusions
As described in this RCT review, the issue of GnRH analogs is still highly debatable in the literature with conflicting evidence and heterogeneous data.
The reason behind the heterogeneity of data may arise from the lack of a consistent uniform definition of premature ovarian failure following chemotherapy, with a clear focus on the resumption of menses as a primary indicator of resumed ovarian function. Focus on pregnancy outcomes as well as survival rates were never the primary outcomes in any of the studies which looked at the role of the use of GnRH analogs in ovarian function preservation. In the above most recent meta-analysis [24], there was an inclusion of RCTs with small patient populations that may not reflect back properly on the general population and may not be strong enough to influence any change in recent guidelines and consensus about using GnRH analogs in young patients receiving chemotherapy.
The administration of GnRH analogs in patients receiving chemotherapy offers a more accessible option for patients and can be used in conjunction with traditional fertility preservation techniques; it also offers feasibility in regard to cost, timing issues, and the need for a partner.
The American Society of Clinical Oncology Clinical Practice Guidelines (2013) regard sperm and embryo cryopreservation as well as oocyte cryopreservation as standard practice with other fertility preservation methods, including the use of GnRH analogs, considered investigational and should be performed by providers with the necessary expertise. Yet, in 2015, the St. Gallen International Expert Consensus panel and the National Comprehensive Cancer Network (NCCN) guidelines have been updated to acknowledge the role of luteinizing hormone-releasing hormone agonists (LHRHa) in preventing chemotherapy-induced POF of hormone receptor-negative breast cancer.
Moreover, in hormone receptor-positive breast cancer patients, the issue of the concurrent use of endocrine therapy, namely tamoxifen along with chemotherapy has demonstrated to be of inferiority in regard to disease-free survival when compared to a sequential administration [30] due to potential antagonism between tamoxifen and the cytotoxic agent. Yet, recently reported excellent survival results with triptorelin administered concurrently with chemotherapy in the Tamoxifen and Exemestane Trial [36] suggests that the use of GnRH agonist may also have a role in improving survival. This overall reflects a hesitant opinion to recommend this technique.