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The Oncologist, Vol. 12, No. 9, 1044-1054, September 2007; doi:10.1634/theoncologist.12-9-1044
© 2007 AlphaMed Press

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Breast Cancer

How to Preserve Fertility in Young Women Exposed to Chemotherapy? The Role of GnRH Agonist Cotreatment in Addition to Cryopreservation of Embrya, Oocytes, or Ovaries

Zeev Blumenfeld

Department of Obstetrics and Gynecology, Rambam Medical Center, Technion-Faculty of Medicine, Haifa, Israel

Key Words. Fertility preservation • Chemotherapy • Gonadotoxicity • GnRH-agonist • Premature ovarian failure

Correspondence: Zeev Blumenfeld, M.D., Department of Obstetrics and Gynecology, Rambam Medical Center, Technion-Faculty of Medicine, Haifa 31096, Israel. Telephone: 972-4-8542577; Fax: 972-4-8543746; e-mail: bzeev{at}techunix.technion.ac.il, z_blumenfeld{at}rambam.health.gov.il

Received December 13, 2006; accepted for publication July 20, 2007.

Disclosure: No potential conflicts of interest were reported by the authors, planners, reviewers, or staff managers of this article.


   Learning Objectives
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 Learning Objectives
Abstract
 Background
 GnRH Agonist Treatment in...
 GnRH Agonist Treatment in...
 Potential Mechanisms of...
 GnRH Agonists as an...
 Addendum
 References
 
After completing this course, the reader will be able to:

  1. Discuss the possibilities for preserving fertility in women exposed to chemotherapy.
  2. List the possible mechanisms put forward to explain the beneficial effect of GnRH agonists in minimizing the gonadotoxic effect of chemotherapy, in particular that of alkylating agents.
  3. Identify the advantages and possible risks and shortcomings of each of the proposed methods for fertility preservation in women exposed to gonadotoxic chemotherapy.
  4. Discuss the possibility of combining several methods to maximize the chances of fertility preservation in these patients.
  5. Explain the gender differences between male and female patients regarding the methods of fertility preservation.

Access and take the CME test online and receive 1 AMA PRA Category 1 CreditTM at CME.TheOncologist.com


   ABSTRACT
Top
 Learning Objectives
 Abstract
Background
 GnRH Agonist Treatment in...
 GnRH Agonist Treatment in...
 Potential Mechanisms of...
 GnRH Agonists as an...
 Addendum
 References
 
The possibilities to preserve fertility in women exposed to chemotherapy are: in vitro fertilization plus embryo cryopreservation, ovarian cryopreservation, unfertilized ova cryopreservation, and the administration of a gonadotropin-releasing hormone (GnRH) agonist. Because none of these methods is ideal, combination of several methods should be considered. Because the chances of preserving gonadal function following combined-modality treatment are significantly better for girls than for boys, simulation of a prepubertal milieu was applied only to women of reproductive age. The administration of GnRH agonists to women with Hodgkin's disease, breast cancer, and other malignancies, or to patients with lupus nephropathy, in parallel with chemotherapy, by others and by us, has demonstrated a significantly lower rate of premature ovarian failure in survivors than in nonrandomized controls. Several prospective, randomized studies are ongoing. A recent meta-analysis found that the administration of a GnRH agonist, in addition to chemotherapy, to patients with breast cancer was associated with less recurrence and superior survival. Several possibilities to explain the beneficial effect of GnRH agonists to minimize chemotherapy-associated gonadotoxicity are suggested: (a) The hypogonadotropic milieu decreases the number of primordial follicles entering the differentiation stage, which is more vulnerable to chemotherapy; (b) The hypoestrogenic state decreases ovarian perfusion and delivery of chemotherapy to the ovaries; (c) A direct effect of the GnRH agonist on the ovary occurs independently of the gonadotropin level; (d) GnRH agonists may upregulate an intragonadal antiapoptotic molecule such as sphingosine-1-phosphate; (e) The GnRH agonist may protect ovarian germline stem cells.


   BACKGROUND
Top
 Learning Objectives
 Abstract
 Background
GnRH Agonist Treatment in...
 GnRH Agonist Treatment in...
 Potential Mechanisms of...
 GnRH Agonists as an...
 Addendum
 References
 
There are several possibilities to preserve future fertility in women exposed to chemotherapy: in vitro fertilization (IVF) and embryo cryopreservation, ovarian tissue cryopreservation, unfertilized ova cryopreservation, and the administration of a gonadotropin-releasing hormone (GnRH) agonist [1–7]; see Table 1. The possibility of administering an adjuvant treatment that may minimize the gonadal damage caused by an otherwise successful treatment program is obviously very attractive [1–7]. Glode et al. [8] tested this hypothesis using a murine model and concluded that an agonistic analogue of GnRH appeared to protect male mice from the gonadal damage normally produced by cyclophosphamide [1]. Although previous suggestions have been made [1, 9] claiming that primordial germ cells fare better than germ cells that are part of an active cell cycle, this hypothesis has not been seriously tested clinically, until recently [1, 4, 7, 10–12]. Whereas several investigators have demonstrated that GnRH agonists inhibit chemotherapy-induced ovarian follicular depletion in the rat [8], uncertainty remains regarding human application [1, 4, 5–12]. The human ovary has lower concentrations of ovarian GnRH receptors and may not necessarily exhibit the same response as the rat ovary [1, 4, 5–12]. Ataya et al. [12], in the only prospective, randomized study performed up to now in primates where follicles were histologically counted [12], found that GnRH agonists protected the ovary against cyclophosphamide-induced damage in rhesus monkeys by significantly decreasing the number of follicles lost during the chemotherapeutic insult, and by decreasing the daily rate of follicular decline.


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  		Table 1. Advantages and disadvantages of fertility-preserving strategies

 
A long-term follow-up of 240 children, 15 years of age or younger, treated using mechlorethamine, vinblastine, procarbazine, and prednisone (MOPP) chemotherapy for Hodgkin's disease (HD) showed azoospermia in 83% of the boys, whereas only 13% of the girls suffered premature ovarian failure (POF) [13]. The chances of preserving gonadal function following combined-modality treatment are significantly better for girls than for boys [1, 4, 7, 13, 14]. In contradiction to the results reported in adults, MOPP chemotherapy in prepubertal girls with HD did not result in POF [15]. Because ovarian function was preserved in most long-term survivors who were treated prepubertally for lymphoma [4, 7, 13–16], but only in about half of the similarly treated adult patients [14, 16], it was clinically logical and therefore tempting to create a temporary prepubertal milieu in women of reproductive age before and during the chemotherapeutic insult [1, 4, 7, 14, 16].


   GNRH AGONIST TREATMENT IN CANCER PATIENTS
Top
 Learning Objectives
 Abstract
 Background
 GnRH Agonist Treatment in...
GnRH Agonist Treatment in...
 Potential Mechanisms of...
 GnRH Agonists as an...
 Addendum
 References
 
We have administered a monthly depot i.m. injection of a GnRH agonistic analogue (D-TRP6-GnRH-a, Decapeptyl C.R., 3.75 mg, Ferring AG, Kiel, Germany) to >125 young patients exposed to gonadotoxic chemotherapy for malignant or nonmalignant diseases, after informed consent, starting before chemotherapy for up to 6 months, in parallel with and until the end of chemotherapeutic treatment [1, 4, 7, 10, 11, 14, 16–19]. The study was approved by the institutional ethics (Helsinki) committee [17]. As previously described in detail [17], the GnRH agonist was offered to every referred female patient before chemotherapy [17]. Those who either did not accept the offer or were referred after the commencement of chemotherapy were included in the control group. In the study group, <7% of patients developed irreversible hypergonadotropic amenorrhea. The remaining patients (>93%) resumed cyclic ovarian function, and 33 patients spontaneously conceived 46 times [1, 4, 7, 10, 11, 14, 16, 17]. These patients were compared with a control group of >125 patients of equivalent age (15–40 years), who were similarly treated with chemotherapy but without the GnRH agonist adjuvant [1, 4, 7, 10, 11, 14, 16–19]. These control group patients were either not referred early enough before starting chemotherapy (in most cases) or were historical controls, treated in the few years before starting our GnRH agonist clinical protocol [17]. Neither the age nor the diagnosis (ratio between HD and non-Hodgkin's lymphoma) differed between the two groups [1, 4, 7, 10, 11, 14, 16–19]. Similar doses of radiotherapy exposure and ratios of patients treated with radiotherapy in addition to chemotherapy were experienced by the two groups [17]. Moreover, the cumulative dose of each chemotherapeutic agent and the mean or median radiotherapy exposure did not differ between the groups [17]. The only significant difference was the rate of POF: <7% in the GnRH agonist cotreatment group versus >53% in the control (chemotherapy without the GnRH agonist) group [1, 4, 7, 10, 11, 14, 16–19]. The relatively advanced age (35, 37, 40, and 40 years) of four of the five patients in the study group who developed POF (the fifth had oophorectomy for pelvic recurrence) suggests that minimizing follicular loss may be efficient only in young patients whose follicular reserve is above a certain limit. In patients older than 36, this reserve may not be sufficient. Indeed, the follicular reserve at the age of 37 is estimated to be around 25,000 and the rate of decrease is accelerated [20]. In keeping with the observation of Ataya et al. [12] that GnRH agonists significantly decrease cyclophosphamide-associated follicular loss, but do not eliminate it completely, it is understandable that the beneficial effect of GnRH agonists is age limited. Therefore, for aggressive chemotherapy protocols, such as escalated bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone (BEACOPP) chemotherapy, we should consider limiting GnRH agonists to patients aged <37.

Similarly, Sklar et al., [21] recently found that survivors of childhood cancer have an 8% risk of suffering POF before the age of 40, compared with <1% in the general population. This is in keeping with our and others' results whereby women of reproductive age receiving a GnRH agonist in addition to chemotherapy suffer POF in about 7%–10% of cases (simulating prepubertal exposure), whereas those treated without the agonist have about a 40% risk for POF [1–7, 10,11,14,16–19, 22, 23].

As opposed to young girls, most prepubertal boys receiving chemotherapy and radiotherapy suffer azoospermia; therefore, there is little rationale to expect a significant benefit from a similar GnRH agonist cotreatment in men [1, 4, 7, 10, 11, 14, 16–19]. In contrast to the encouraging protective effect of GnRH agonist cotreatment with chemotherapy, no protection from ovarian damage caused by irradiation to rats could be provided by the GnRH agonist [1, 7, 14, 16].

Similar experience and results regarding the protective effect of a GnRH agonist against chemotherapy-associated gonadotoxicity were recently reported in adolescent female patients [22]. Whereas all the GnRH agonist–treated patients resumed cyclic ovarian function, all patients in the chemotherapy-alone (without a GnRH agonist) group experienced hypergonadotropic amenorrhea in spite of their young, adolescent age [22]. Similarly, Castelo-Branco et al. [23] reported a 10% rate of POF in their GnRH agonist–treated hemato-oncologic female patients, compared with a 77% POF rate in their control group.

Similarly, a GnRH agonist prevented chemotherapy-associated POF in premenopausal breast cancer patients [24–29]. All 13 patients in one study [24], aged 26–39 years, resumed normal ovarian function after a mean of 4.9 months postchemotherapy, and in another study [25], 86% of the 64 premenopausal patients (27–50 years of age) resumed cyclic menstruation, despite a relatively advanced median age of 42 years. In a recent update of their previous study [25], Recchia et al. [26] found that all their breast cancer patients younger than 40 who received GnRH agonist cotreatment in addition to chemotherapy resumed cyclic ovarian function, with excellent 5- and 10-year survival rates. In breast cancer, at least four phase II studies evaluated the activity of ovarian suppression with GnRH agonists in preserving fertility and ovarian function [24–29]. Those studies [24–29] have clearly demonstrated that GnRH agonist cotreatment enables the resumption of ovarian function in a high percentage of treated patients, in the range of 83%–96% [28]. A few ongoing, phase III, randomized controlled trials (such as the Southwest Oncology Group led U.S. Intergroup Trial S0230, which also has the participation of the International Breast Cancer Study Group) will probably be able to unequivocally answer the debated question regarding the role of GnRH agonists in preserving ovarian function and fertility in young premenopausal patients exposed to gonadotoxic chemotherapy [29–31].

Although pregnancies occurred in both groups in our series, it should be emphasized that, whereas all the 22 pregnancies in the "control" group occurred in patients aged 16–26 years when exposed to chemotherapy, the ages of the 33 patients who conceived 46 times in the GnRH agonist/chemotherapy cotreatment were 18–33 years at exposure to chemotherapy [1, 4, 7, 10, 11, 14, 16–19]. This difference may suggest a possible prolongation of the "fertility window" by 7 years (or more) using GnRH agonist adjuvant cotreatment in parallel with chemotherapy. This is in keeping with the experience reported by others [32, 33] who also found the median age of pregnant survivors of lymphomas to be 18 years (range, 13–28), with only few reported pregnancies for female patients >30 years old.

If this protective effect observed in our and others' recent preliminary studies of GnRH agonists and chemotherapy on future ovarian function is confirmed in larger and prospective randomized studies, which are currently ongoing in Italy [29], the U.S. [30], and Germany [34], for breast cancer [29, 30], HD (German Hodgkin's Lymphoma Study Group), and lupus erythematosus patients [34] receiving cyclophosphamide pulsatile therapy, it may become mandatory to use this cotreatment protocol in every young woman undergoing chemotherapy. Thus, ovarian protection may enable the preservation of future fertility in survivors and, in addition, prevent the bone demineralization and osteoporosis associated with hypoestrogenism and ovarian failure, and the menorrhagia with resultant anemia associated with chemotherapy-induced thrombocytopenia [1, 4, 7, 10, 11, 14, 16–19, 34–37].

Notwithstanding all the above, two studies published in the New England Journal of Medicine [38, 39] reported that good observational studies give results similar to those of randomized controlled trials. The first [38] "found little evidence that estimates of treatment effects in observational studies reported after 1984 are either consistently larger than or quantitatively different from those obtained in randomized, controlled trials." Moreover, the second [39] concluded that "the results of well-designed observational studies, do not systematically overestimate the magnitude of the effects of treatment as compared with those in randomized, controlled trials on the same topics."

It has been suggested that future studies should examine GnRH antagonists instead of agonists for the achievement of a faster pituitary–ovarian desensitization, eliminating the waiting period of 7–14 days needed by the GnRH agonist to achieve downregulation [4, 7, 10, 11, 14, 16–18]. However, a recent study [40] has concluded that, in contrast to the "well known effects of GnRH agonists to reduce chemotherapeutic destruction of primordial follicles, GnRH antagonists do not protect the ovary from the damaging effects of cyclophosphamide." In that study [40], administration of cyclophosphamide to adult mice caused a nearly 50% reduction in the number of primordial follicles. The GnRH agonist leuprolide (Lupron®; TAP Pharmaceuticals, Lake Forest, IL) alone had no effect on the number of primordial follicles, but significantly minimized the follicular depletion caused by cyclophosphamide when coadministered [40]. In contrast, the GnRH antagonist antide did not prevent the depletion of primordial follicles caused by cyclophosphamide. Surprisingly, both tested antagonists, antide and cetrorelix, caused a significant reduction in the number of primordial follicles (even without cyclophosphamide) (p < .05) [40]. This observation, although preliminary, casts significant doubt on the assumption that GnRH antagonists may possibly be substituted for agonists in the future for minimizing chemotherapy-associated gonadotoxicity. Furthermore, this preliminary study may possibly explain the tendency toward lower pregnancy rates in assisted-reproduction technique/IVF cycles using antagonists versus agonists [41].


   GNRH AGONIST TREATMENT IN OTHER DISEASES
Top
 Learning Objectives
 Abstract
 Background
 GnRH Agonist Treatment in...
 GnRH Agonist Treatment in...
Potential Mechanisms of...
 GnRH Agonists as an...
 Addendum
 References
 
GnRH agonist cotreatment may also be applied to young women receiving cytotoxic chemotherapy for noncancerous, benign diseases. Because 25%–50% of young women with systemic lupus erythematosus of reproductive age may develop POF after cyclophosphamide therapy [1, 14, 18, 34, 42, 43], GnRH agonist cotreatment may possibly be coadministered to these young women in parallel with the cytotoxic treatment, as well as to any woman with an autoimmune disorder treated with gonadotoxic chemotherapy, mainly alkylating agents [1, 14, 18, 34, 42, 43]. Indeed, the results of our group and those of Somers et al. [43] support the effectiveness of GnRH agonist administration to patients receiving cyclophosphamide pulses for autoimmune diseases [1, 14, 18, 34, 42, 43].


   POTENTIAL MECHANISMS OF GONADOTOXIC PROTECTION
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 Learning Objectives
 Abstract
 Background
 GnRH Agonist Treatment in...
 GnRH Agonist Treatment in...
 Potential Mechanisms of...
GnRH Agonists as an...
 Addendum
 References
 
How can we possibly explain the beneficial effect of GnRH agonists for minimizing the gonadotoxic effect of chemotherapy, in particular that of alkylating agents? Several explanations may be put forward.

I: Interruption of Follicle-Stimulating Hormone Secretion
One can conceivably hypothesize that alkylating agents may bring about an increased rate of destruction/apoptosis of nonresting follicles, and subsequently a decrease in the secretion of sex steroids and inhibins produced by these follicles at different stages of maturation and differentiation. The resultant decrease in sex steroid and inhibin secretion will decrease their plasma concentrations and subsequently the negative feedback on the hypothalamus and pituitary, resulting in an increase in follicle-stimulating hormone (FSH) secretion. The increased FSH secretion may bring about an increased recruitment of preantral follicles to enter the one-way differentiational path of maturation, being further exposed to the gonadotoxic effects of the alkylating agents, ending in an increased, exponential rate of follicular apoptosis and degeneration. This unfortunate vicious cycle may be interrupted by the GnRH agonist administration through its ability to prevent the increase in FSH concentrations [6] (Fig. 1).


Figure 1
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  		Figure 1. A suggested pathophysiologic mechanism of chemotherapy-induced gonadotoxicity. GnRH-a administration may rescue the follicles from accelerated atresia by preventing the increased FSH concentration induced by the negative feedback of low sex steroids and inhibin levels.

Abbreviations: E2, estradiol; FSH, follicle-stimulating hormone; GnRH-a, gonadotropin-releasing hormone agonist.

 
Indeed, supporting the possible detrimental effect of high gonadotropin concentrations on primordial and primary follicles, it has been demonstrated [44] that transgenic mice with increased luteinizing hormone (LH) concentrations that have a similar number of follicles as wild-type controls at birth suffer a significant premature loss of their primordial and primary follicles, in keeping with our suggested pathophysiologic hypothesis.

Moreover, others [45–47] have shown that primordial and primary follicles may express mRNA for FSH and LH receptors, in keeping with our suggested vicious cycle pathophysiologic mechanism and with the notion that even primordial and primary follicles may be gonadotropin dependent, although indirectly [48], in contrast to the previous theory claiming that they are totally gonadotropin independent. Even if we assume that the primordial follicles are gonadotropin independent, because no gonadotropin receptors have been demonstrated on their primordial granulosa cells (GCs), they are dependent on growth factors (GFs) such as bone morphogenic protein (BMP)-4, BMP-7, and BMP-9 and activin [48], which are secreted by the more advanced, preantral follicles, beyond the secondary stage, and these secreted GFs induce the maturation of primordial follicles [48]. The more mature, preantral follicles, beyond the secondary stage, secrete these GFs in response to FSH stimulation [48]. Pituitary desensitization, induced by GnRH agonist administration, prevents the secretion of GFs 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 [1, 12, 14, 36, 48, 49]. Supporting this hypothesis is the recent demonstration [50] that gonadotropins may have an apoptotic effect on the theca-interstitial cells of isolated rat follicles in vitro. This group [50] has shown 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 [50]. Furthermore, Adriaens et al. [51] have shown that differential FSH exposure in preantral follicle culture has marked effects on folliculogenesis and oocyte developmental competence. Although early preantral follicles are independent of FSH for their initial growth, FSH receptors are present on the GCs of these follicles [51]. The best known FSH receptor is FSHR-1 [51]. However, several events occur well before the expression of the stimulatory G-protein–coupled FSHR-1 [51], suggesting that other FSHR motifs might function to play a role during development. It was proven that a new splice variant, FSHR-3, is present in immature ovaries and that there is a positive correlation between follicle differentiation and the expression of this receptor [51, 52]. In the human, one third of the primary and two-layered follicles produce FSHR mRNA and all multilaminar follicles are positive for FSHR mRNA [46, 47]. Human ovarian xenografts in nude and hypogonadal mice contain follicles progressed beyond the secondary stage only when animals are treated with FSH [53]. These observations are in contrast to the older assumption that early follicles are completely gonadotropin independent [51]. Furthermore, two recent publications found that the proliferation of primordial germ cells is gonadotropin dependent and is mediated by Akt/phosphatase and tensin homologue deleted on chromosome ten (PTEN) signaling [54, 55]. High concentrations of estrogens stimulate mouse primordial germ cell growth in vitro through the somatic cells of the gonad [54, 55]. Moreover, estrogens stimulate the transcription of the Steel gene and the production of c-Kit ligand in gonadal somatic cells, and this GF is likely to be responsible for the observed stimulation of primordial germ cell growth via an Akt/PTEN pathway [54, 55]. 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 [56]. Therefore, the older assumption that early follicles are Gn independent may need re-evaluation and reassessment [47–52, 54–56].

II: Decrease in Utero-Ovarian Perfusion
Another possible explanatory mechanism for the beneficial effect of GnRH agonists on decreasing chemotherapy-associated gonadotoxicity is the decrease in utero-ovarian perfusion resulting from the hypoestrogenic state generated by pituitary–gonadal desensitization [57, 58]. High estrogen concentrations significantly increased ovarian perfusion and vessel endothelial area in a rat model of ovarian hyperstimulation, and this effect was significantly and dose-dependently inhibited by administration of a GnRH agonist [58]. The decreased utero-ovarian perfusion induced by the GnRH agonist may result in a lower total cumulative exposure of the ovaries to the chemotherapeutic agents as compared with a "control" patient, in a normoestrogenic milieu, thus resulting in less gonadotoxicity.

III: Activation of GnRH Receptors
It has been shown that not only rodent but also primate and human gonads contain GnRH receptors [1, 59–61]. In an ovarian carcinoma cell line, Grundker and Emons [60] have shown that 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 GC, or possibly on another ovarian compartment in addition to its possible hypogonadotropic effect, is an open question of significant interest. Additional studies are obviously necessary to answer this important question. Most recently, proof of a direct effect of GnRH agonists, independent of the hypogonadotropic milieu, was provided by Imai et al. [62]. Those investigators [62] have shown direct in vitro protection from doxorubicin-induced GC damage by a GnRH agonist.

IV: Upregulation of Sphingosine-1-Phosphate
Another possibility is that GnRH agonists may upregulate an intragonadal antiapoptotic molecule such as sphingosine-1-phosphate (S1P). Tilly et al. [63–65] have identified several molecules that are required for chemotherapy-induced oocyte apoptosis. While much of their work has relied on gene knockout mice, they have identified a small lipid antagonist of the proapoptotic second messenger ceramide, S1P, as a potent protective molecule [63–65].

Mouse oocytes that were exposed to doxorubicin did not degenerate in a pathological fashion, but initiated programmed cell death [65]. Targeted disruption of the Bax gene in mice or, more recently, targeted expression of the Bax antagonist Bcl-2 to the female mouse germ line may protect the oocytes from the gonadotoxic effect of doxorubicin [66, 67]. Unfortunately, comparable genetic technologies for protection against chemotherapy-induced gonadotoxicity are not yet feasible in humans [4].

Experiments in mice tested the possibility that S1P could be administered in vivo to protect the ovaries from radiotherapy-induced damage [64]. Suppression of caspase activity in models of Bax-driven apoptosis eventually activated a default pathway of cell death, which is more akin to primary necrosis, probably because the mitochondria are still "damaged" by Bax in a caspase-independent manner [68]. Whereas oocyte death induced by chemotherapy is Bax dependent [65], caspase inhibition was excluded as a possibility. Therefore, a preceding step in the oocyte death program was elected—the proapoptopic molecule ceramide.

Many somatic cell types generate ceramide, by either synthetic or hydrolytic mechanisms, in response to radio- or chemotherapy [69]. Ceramide may function in several stages of the programmed cell-death pathway, such as "capping" of death receptors [70] and helping Bax to insert into mitochondrial membranes [71]. In hematopoietic cells, S1P counteracts ceramide proapoptotic effects [72, 73]. The S1P molecule can also prevent doxorubicin-induced oocyte death in vitro [64, 65]. Furthermore, oocytes that lack acid sphingomyelinase—a hydrolytic enzyme that generates ceramide [69]—are resistant to doxorubicin-induced apoptosis in vitro [64]. Young adult female mice were given a single injection of S1P into the bursal cavity, which surrounds each ovary [64, 74]. Two hours later, they were irradiated with an amount sufficient to destroy the majority of the primordial oocyte reserve. Two weeks after irradiation, the ovaries were analyzed. No differences were observed between mice that had not been irradiated and those that had been protected by S1P in vivo before irradiation. In contrast, irradiated mice that did not receive S1P suffered a pronounced loss of oocytes and reduced embryonic developmental potential of the remaining oocytes [64, 74]. With preliminary data from in vivo mating trials supporting the conclusions that S1P preserves a normal level of fertility in female mice that are exposed to anticancer therapy [74, 75], minimizing the gonadal toxicity of such treatments in female cancer patients might one day prove feasible [1, 11, 63, 73–76].

More recently, it was demonstrated, for the first time, that S1P-based protection of the female germ line from radiation is not associated with discernible propagation of genomic damage at the anatomical, histological, biochemical, or cytogenetic level [75]. Moreover, this investigation [75] provides the first credible prospective evidence that preservation of ovarian function and fertility postirradiation can be safely and effectively achieved in vivo, using S1P-based strategies.

Whether the GnRH agonist adjuvant cotreatment positive effect is direct or possibly associated with an intraovarian increase in S1P is a question of tremendous interest and clinical impact. It obviously awaits further investigation [76].

V: Protection of Undifferentiated Germ Line Stem Cells
Most recently, Johnson et al. [77] presented revolutionary data whereby mouse ovaries may possess mitotically active germ cells that continuously replenish the pool of immature follicles. These germ line stem cells (GSCs) may exist in the mouse ovary and regenerate the primordial follicle pool. Their observations contradict the basic doctrine of reproductive biology, whereby most mammalian females lose the capacity for germ-cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth.

Exposure of prepubertal female mice to the mitotic germ-cell toxicant busulphan eliminated the primordial follicle reserve by early adulthood without inducing atresia [77]. These data may establish the existence of undifferentiated proliferative germ cells in the female gonad, similar to the situation in the male, that sustain oocyte and follicle production in the postnatal mammalian ovary [77]. One may speculate that the GnRH agonist protective effect may possibly be through protection of the undifferentiated GSCs, which ultimately generate de novo primordial follicles. Indeed, the observation of temporary, high, reversible FSH concentrations in one third of our patients several months after the chemotherapy and GnRH agonist cotreatment, even in those who spontaneously conceived later on, may point toward reversible gonadotoxicity. The possible de novo formation of follicles by the surviving GSCs brings about a decrease in FSH concentration and return of regular cycles, ovulation, and even gestation.


   GNRH AGONISTS AS AN ADJUNCT TO CURRENT FERTILITY-PRESERVING STRATEGIES
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 Learning Objectives
 Abstract
 Background
 GnRH Agonist Treatment in...
 GnRH Agonist Treatment in...
 Potential Mechanisms of...
 GnRH Agonists as an...
Addendum
 References
 
The IVF procedure is a clinically accepted resolution for fertility preservation in young women before chemotherapy or radiotherapy for breast cancer or other malignancies. However, ovulation induction with gonadotropins may increase the levels of sex steroids with possible aggravation of an estrogen receptor–positive or progesterone receptor–positive breast neoplasm. Therefore, it was recently recommended that aromatase inhibitors be used in the protocols of controlled ovarian hyperstimulation (COH) in breast cancer patients before IVF [78]. The American Society of Clinical Oncology (ASCO) states that "for women with hormone-sensitive tumors (such as breast cancer) alternative hormonal stimulation approaches such as letrozole or tamoxifen have been developed to theoretically reduce the potential risk of estrogen exposure" [79]. However, concerns regarding safety, for both women with breast cancer and babies born after this type of ovarian stimulation, have recently been published [29]. Del Mastro and Venturini [29] claimed that, because of potential biases, comparison of short-term breast cancer recurrence rates between patients who have undergone ovarian stimulation and nonrandomly assigned controls cannot be considered as a demonstration of the safety of the procedure for women with breast cancer [78, 79]. Regarding safety for the embryo, recent data indicate that babies born after treatment with letrozole for ovarian stimulation may have a high risk for locomotor and cardiac malformations [80]. Taking into account these concerns, the use of ovarian stimulation with letrozole should be considered experimental and potential risks disclosed to patients. Furthermore, Del Mastro and Venturini [29] suggested that this protocol, using letrozole for COH for IVF, should be discouraged instead of being considered as a potential option for women with breast cancer, as indicated in ASCO recommendations [79].

It is agreed by all that a large, prospective, randomized study is needed to unequivocally substantiate the role of GnRH agonists as a possible effective adjunct to minimize chemotherapy-associated gonadotoxicity. Indeed, several such prospective, randomized studies are presently ongoing: the Zoladex Rescue of Ovarian Function (ZORO) study in Germany, an Italian multicenter study for breast cancer patients, the Southwest Oncology Group led U.S. Intergroup trial S0230 (which also has participation of the International Breast Cancer Study Group) [30], the German Hodgkin's Lymphoma Study Group multicenter study, a U.K. lymphoma multicenter study, and a Spanish Lymphoma multicenter study. If these studies come up with unequivocal results demonstrating the beneficial effect of GnRH agonists in minimizing chemotherapy-associated gonadotoxicity, the categorical statement of Sonmezer and Oktay [81] against GnRH agonist cotreatment may do harm to many patients who could benefit from such a clinical combination, in addition to IVF and ovarian tissue cryopreservation, while waiting for the results of these prospective studies in humans.

If GnRH agonist cotreatment proves to be of no value, its use will not have done any harm, because it is presently recommended in addition to IVF and embryo cryopreservation and/or ova or ovarian tissue cryopreservation. Furthermore, it may decrease thrombocytopenia-associated menorrhagia, as recently described [37]. Moreover, the recent studies of Recchia et al. [25, 26] and Del Mastro et al. [27] supporting this cotreatment in breast cancer have shown that, in premenopausal women with early breast carcinoma, the addition of a GnRH agonist to adjuvant therapy and temporary total estrogen suppression in patients with estrogen receptor–positive disease was well tolerated, protected long-term ovarian function, and appeared to improve the expected clinical outcome [25, 26]. After a median follow-up of 75 months, normal menses resumed in all patients <40 years of age and in 56% of patients >40 years old [25, 26]. The projected recurrence-free survival rates at 5 years and 10 years were 84% and 76%, respectively, and the projected overall survival rates at 5 years and 10 years were 96% and 91%, respectively [25, 26], contradicting the speculations that GnRH agonists may decrease the effect of chemotherapy on malignant cells [81]. In the study by Del Mastro et al. [27], menses resumption was observed in 16 of 17 patients (94%) <40 years of age and in five of 12 patients (42%) >40 years old. Those authors concluded that goserelin given before and during chemotherapy may prevent premature menopause in the majority of patients. These results [25–27] corroborate the findings of other studies in breast cancer patients, showing that the activity of GnRH agonists in preventing early menopause is in the range of 86%–100% [25–29]. Using cyclophosphamide, epirubicin, and fluorouracil regimens like that used in the study of Del Mastro et al. [27], the expected rate of menstrual activity resumption is nearly 40% in the overall group of premenopausal patients, 64% in patients <40 years old, and 30% in those aged ≥40. These studies [25–29] suggest a significant improvement in the preservation of ovarian function by the use of GnRH agonists in keeping with our studies in hematologic malignancies [1,4, 7, 10,11,14,16–19, 49]. Sonmezer and Oktay [81] claim that they do not accept the published studies on GnRH agonist cotreatment because they do not include a prospective, randomized, control group. Their studies on IVF and those on ovarian cryopreservation did not either. Sonmezer and Oktay [81] erroneously cite a study by Waxman et al. [82] from 1987 as proof of negative results, although it was conducted mainly in men, whereas we clearly pointed out that GnRH agonists are effective only in women and ineffective in men [1,4, 7, 10,11,14,16–19, 49]. Waxman et al. [82] treated only eight women (and 20 men) with buserelin. There was not enough power to show a difference between the GnRH agonist–treated group of women (4 of 8 patients with POF) and the controls (6 of 9 with POF) [82]. Furthermore, in a later publication [83], Waxman himself refers to his previous study, suggesting that the used analogue might have been inappropriate: "Unfortunately, a randomized trial has shown no protection, but the wrong analogue regimen may have been used."

Most recently, Burns et al. [84] have found that parents and female adolescents are interested in options to help preserve fertility during cancer treatment, but they are not willing to postpone treatment for this purpose. For those patients who refuse postponement for IVF [84], a GnRH agonist may be an alternative option. Failure to offer this option may be considered as a possible conflict of interest for those commercial centers that are interested in the income from IVF.

Because most of the methods involving ovarian or egg cryopreservation are not yet clinically established and successful, one should be very careful to provide these young patients with all the information concerning the various modalities to minimize gonadal damage and preserve ovarian activity and future fertility [49]. Furthermore, combining the various modalities for a specific patient may increase the odds of preservation of future fertility. There is no contraindication to ovarian biopsy for cryopreservation combined with GnRH agonist administration and follicular aspiration for IVF and embryo freezing where the patient has a spouse/partner. In cases where the chemotherapy has caused POF, as is frequently the case in total body irradiation and bone marrow transplantation, the patient has cryopreserved primordial follicles and/or frozen embryos to fall back upon. However, in cases where conventional chemotherapy regimens such as those commonly used for young lymphoma patients are applied, GnRH agonist cotreatment may preserve ovarian function without necessitating the use of cryopreserved ova or embryos.

Future investigation is needed to address the safety and efficacy of oocytes, follicles, or ovary cryopreservation, and the most efficient way of using the cryopreserved, thawed tissue. The results of multicenter, prospective, randomized studies are awaited to substantiate the in vivo ef-fect of GnRH agonists as an unequivocal means for minimizing follicular apoptosis.


   ADDENDUM
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 Learning Objectives
 Abstract
 Background
 GnRH Agonist Treatment in...
 GnRH Agonist Treatment in...
 Potential Mechanisms of...
 GnRH Agonists as an...
 Addendum
References
 
A recent meta-analysis published in the Lancet [85], based on data from 11,906 premenopausal women with early breast cancer randomized in 16 trials, has concluded that the addition of luteinizing-hormone-releasing hormone agonists to tamoxifen, chemotherapy, or both reduced recurrence by 12.7% (95% confidence interval, 2.4%–21.9%; p = .02) and death after recurrence by 15.1% (95% confidence interval, 1.8%–26.7%; p = .03). This contradicts the previously raised hypothetical speculation that GnRH agonists may decrease the efficacy of chemotherapy in ER positive breast cancer.


   REFERENCES
Top
 Learning Objectives
 Abstract
 Background
 GnRH Agonist Treatment in...
 GnRH Agonist Treatment in...
 Potential Mechanisms of...
 GnRH Agonists as an...
 Addendum
 References
 

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