‘Ovarian cryopreservation versus ovarian suppression by GnRH analogues: primum non nocere’: Reply

Kutluk Oktay1, Murat Sonmezer2 and Ozgur Oktem1

1 Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University, New York, USA 2 Ankara University School of Medicine, Department of Obstetrics and Gynecology, Ankara, Turkey

Dear Sir,

We thank Professor Blumenfeld for raising this matter (Blumenfeld, 2004Go). We agree, as we concluded in our debate article, that patients should be presented with all the facts about all applicable options. In our centre, we counsel the patient regarding the uncertainty of the benefit of ovarian suppression, and do not recommend it as a proven beneficial method. In the debate article by Revel and Schenker (2004Go), the conclusion was similar to ours in that there is no consensus regarding the effectiveness of ovarian suppression. Because the benefits and long-term effects of GnRH analogue administration are unknown at the present time, it should only be given as a part of prospective research and under ethics committee-approved protocols. It cannot be assumed that GnRH analogue administration is without drawbacks. One drawback is that patients can be misled ino believing that this is a foolproof approach, and they may decide not to resort to more established approaches such as embryo cryopreservation. Apart from the cost and severe hot flushes, there could also be potential risks to the patient. When given to breast cancer patients prior to chemotherapy, the hypoestrogenic milieu can put cancer cells into the G0 phase, making them less responsive to chemotherapy (Mullen et al., 1991Go; Emons et al., 2003Go). In fact, 50% of breast cancer cases express GnRH receptors, and it has been shown that the stimulation of these receptors by native GnRH and GnRH analogues can lead to protection of cancer cells from chemotherapy-induced apoptosis (Emons et al., 2003Go). Cyclophosphamide, the most potent gonadotoxic agent, has to be broken down into its toxic metabolites before it can exert its chemotherapeutic effects. No study has looked at whether GnRH analogue administration interferes with this process; it is theoretically possible that if there is a decrease in gonadal toxicity, it is due to reduced chemotherapeutic effectiveness. Another theoretical concern is the increased gonadotoxicity. Gonadotrophins induce a series of antioxidant enzymes called glutathione S-transferases (GSTs) (Toft et al., 1997Go). These enzymes are present in granulosa cells of follicles of various stages in the human ovary (Rahilly et al., 1991Go) and play a role in detoxifying chemotherapeutics (Gamcsik et al., 1999Go). Ovarian suppression reduces the expression of these enzymes, in theory rendering follicles more vulnerable to the effects of chemotherapy.

Dr Blumenfeld presented a different interpretation of the study of Teinturier et al. (1998Go). In that report, while 12 of 21 subjects had clinical evidence of gonadal failure, another patient had laboratory evidence. Moreover, two patients had gonadotrophin levels at the upper limit of normal, bringing the percentage of subjects with diminished ovarian reserve to 71%. Interestingly, all 10 patients who received busulfan developed ovarian failure. As shown by Meirow et al. (1999Go) in a rodent model, immediate reproductive endocrine performance is not a sign that ovarian reserve is not altered. Because younger patients have a larger ovarian reserve, and because of the variation in chemotherapy regimens, they may not immediately suffer from ovarian failure. Eventually, every person who receives alkylating chemotherapy will suffer from ovarian failure. Since the median ages when chemotherapy was received and at follow-up were 7 (range 1.2–13 years) and 14.5 (range 11–21 years), respectively, it cannot be concluded that the remaining 29% of patients were not affected by chemotherapy. In fact, the major weakness of previous studies on the role of GnRH analogues has been the short duration of follow-up, compared with controls. In the study by Blumenfeld et al., 16 women with lymphomas who received GnRH analogue co-treatment for gonadal protection were compared with a historical control group. While all patients in the GnRH analogue group resumed menses, 61% in the control group experienced premature ovarian failure. However, mean follow-up was only 1.7 ± 1.0 years (range 0.5–4) in the study patients compared with 7.0 ± 4.9 years (range 1.5–8.0) in the controls (Blumenfeld et al., 1996Go). Moreover, only four of 16 in the study group received cyclophosphamide, the principal gonadotoxic drug, compared with 10 of 18 in the control group. Of concern to us is that two of the patients in the group receiving GnRH analogue did not respond to chemotherapy and died, while all survived in the chemotherapy only group. The latter observation further increases our concern that GnRH analogues may be diminishing the effectiveness of chemotherapy. The report by Pereyra Pacheco et al. (2001Go) was a case series report. They reported a retrospective group of five pre-menarchal children (aged 3–7.5 years) who received polychemotherapy, and descriptively compared them with a ‘prospective’ group of 12 post-menarchal children (aged 14.7–20 years) who received GnRH analogues prior to chemotherapy and another retrospective group of four post-menarchal children (aged 15.9–20 years) who received chemotherapy without GnRH analogue co-treatment The duration of follow-up was different between the pre-menarchal and post-menarchal groups (18 versus 5 and 6 years, respectively). No statistical analysis was done to control for differences in groups or to compare outcomes. Dr Blumenfeld cited the presence of GnRH receptors on ovarian cancer lines as supporting evidence for the presumed gonadal protective effect of GnRH analogues. It is a basic biological fact that cancer cell lines are aberrant and express many genes that are not expressed in normal cells. Besides, the cell lines that were used in the study by Volker et al. (2002Go) were of surface epithelium origin and not germ cells. There is no evidence that human primordial and pre-antral follicles express GnRH receptors.

Blumenfeld argued that there is sufficient evidence for the presence of FSH receptors on human primordial follicles. The presence of mRNA does not mean that functional receptors are expressed on cells, and does not prove a ‘physiological’ role. In the study of Patsoula et al. (2003Go), oocytes were from antral follicles, not from primordial or pre-antral follicles. Also, when using the extremely sensitive technique of RT–PCR, positive results are questionable because of the inevitable contamination from surrounding mature granulosa cells. Moreover, in the study of Zheng et al. (1996Go), which focused on FSH mRNA expression in tubal epithelium, only a weak focal mRNA signal was seen by in situ hybridization in primordial follicles, and the data were not shown. When using in situ hybridization, because of the minute size and flattened shape of primordial pre-granulosa cells, it is technically challenging to distinguish background signals from signals in primordial follicles. By RT–PCR, we did not detect FSH receptor mRNA in isolated human primordial follicles (Oktay et al., 1997Go). Moreover, when xenografted in hypogonadal-immunodeficient mice, human ovarian follicles continue to initiate growth (Oktay et al., 1998Go). In patients with FSH receptor mutation, follicles continue to initiate growth (Meduri et al., 2003Go). There has been no evidence for the presence of FSH receptor protein on primordial follicles. Primordial follicles continue to initiate growth through hypogonadal states such as pregnancy and use of oral contraception, and ovarian suppression by oral contraceptives does not prevent chemotherapy-induced gonadal damage (Whitehead et al., 1983Go). As the authors of the study pointed out, increased depletion of primordial follicles in LH-overexpressing mutant mice does not indicate a direct effect of LH (Flaws et al., 1997Go). In fact, in that study, the authors showed a reduction and not an increase in the fraction of follicles entering the growth pool (primary), and concluded that the effect of LH was indirect, and that their data did not prove that primordial follicles were gonadotrophin responsive. As was discussed in that report, LH receptors have never been detected in primordial or early pre-antral follicles, and the authors explained the loss of primordial follicles by the toxic effects of the local endocrine milieu stimulated by supraphysiologically high LH stimulation on stromal cells.

Animal studies are, by definition, prospective. In the study of Ataya et al., 1995Go, three primates were studied in each of the cyclophosphamide and cyclophosphamide + GnRH analogue groups. One animal in the cytoxan group died prematurely, further reducing the sample size. In the only prospective controlled study in humans, albeit small, no benefit of GnRH analogue co-treatment against chemotherapy-induced gonadal damage could be demonstrated (Waxman et al., 1987Go).

Recent valuable work has challenged the notion that ovarian follicular reserve cannot be renewed in rodents (Johnson et al., 2004Go). The belief that pre-pubertal girls are immune from chemotherapy damage is another old dogma, which gave rise to the hypothesis that ovarian suppression through the use of GnRH analogues should protect the gonads against chemotherapy. We should challenge this dogma by carefully analysing the pre-existing scientific data, and by conducting a prospective randomized study. As has been the case of diethylstilbestrol and HRT, too much confidence in descriptive and retrospective data may result in adverse effects in patients, which will be against the principle of ‘primum non nocere’ (Beauchamp and Childress, 1994Go).

References

Atya K, Rao LV, Lawrence E and Kimmel R (1995) Luteinizing hormone-releasing hormone agonist inhibits cyclophosphamide-induced ovarian follicular depletion in rhesus monkeys. Biol Reprod 52,365–372.[Abstract]

Beauchamp TL and Childress JF (1994) Principles of Biomedical Ethics, 4th edn. Oxford University Press, New York, p. 11.

Blumenfeld Z (2004) Ovarian cryopreservation versus ovarian suppression by GnRH analogues: primum non nocere. Hum Reprod 19,

Blumenfeld Z, Avivi I, Linn S, Epelbaum R, Ben-Shahar M and Haim N (1996) Prevention of irreversible chemotherapy-induced ovarian damage in young women with lymphoma by a gonadotrophin-releasing hormone agonist in parallel to chemotherapy. Hum Reprod 11,1620–1626.[Abstract]

Emons G, Grundker C, Gunthert AR, Westphalen S, Kavanagh J and Verschraegen C. (2003) GnRH antagonists in the treatment of gynecological and breast cancers. Endocr Relat Cancer 10,291–299.[Abstract/Free Full Text]

Flaws JA, Abbud R, Mann RJ, Nilson JH and Hirshfield AN (1997) Chronically elevated luteinizing hormone depletes primordial follicles in the mouse ovary. Biol Reprod 57,1233–1237.[Abstract]

Gamcsik MP, Dolan ME, Andersson BS and Murray D (1999) Mechanisms of resistance to the toxicity of cyclophosphamide. Curr Pharm Des 5,587–605.[ISI][Medline]

Johnson J, Canning J, Kaneko T, Pru JK and Tilly JL (2004) Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428,145–150.[ISI]

Meduri G, Touraine P, Beau I, Lahuna O, Desroches A, Vacher-Lavenu MC, Kuttenn F and Misrahi M (2003) Delayed puberty and primary amenorrhea associated with a novel mutation of the human follicle-stimulating hormone receptor: clinical, histological, and molecular studies. J Clin Endocrinol Metab 88,3491–3498.[Abstract/Free Full Text]

Meirow D, Lewis H, Nugent D and Epstein M (1999) Subclinical depletion of primordial follicular reserve in mice treated with cyclophosphamide: clinical importance and proposed accurate investigative tool. Hum Reprod 14,1903–1907.[Abstract/Free Full Text]

Mullen P, Scott WN and Miller WR (1991) Growth inhibition observed following administration of an LHRH agonist to a clonal variant of the MCF-7 breast cancer cell line is accompanied by an accumulation of cells in the G0/G1 phase of the cell cycle. Br J Cancer 63,930–932.[ISI][Medline]

Oktay K, Briggs D and Gosden RG (1997) Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab 82,3748–3751.[Abstract/Free Full Text]

Oktay K, Newton H, Mullan J and Gosden RG (1998) Development of human primordial follicles to antral stages in SCID/hpg mice stimulated with follicle stimulating hormone. Hum Reprod 13,1133–1138.[Abstract]

Patsoula E, Loutradis D, Drakakis P, Michalas L, Bletsa R and Michalas S (2003) Messenger RNA expression for the follicle-stimulating hormone receptor and luteinizing hormone receptor in human oocytes and preimplantation-stage embryos. Fertil Steril 79,1187–1193.[CrossRef][Medline]

Pereyra Pacheco B, Mendez Ribas JM, Milone G, Fernandez I, Kvicala R, Mila T, Di Noto A, Contreras Ortiz O and Pavlovsky S (2001) Use of GnRH analogs for functional protection of the ovary and preservation of fertility during cancer treatment in adolescents: a preliminary report. Gynecol Oncol 81,391–397.[CrossRef][Medline]

Rahilly M, Carder PJ, al Nafussi A and Harrison DJ (1991) Distribution of glutathione S-transferase isoenzymes in human ovary. J Reprod Fertil 93,303–311.[Abstract]

Revel A and Schenker J (2004) Ovarian tissue banking for cancer patients: is ovarian cortex cryopreservation presently justified? Hum Reprod 19,14–19[Abstract/Free Full Text]

Teinturier C, Hartmann O, Valteau-Couanet D, Benhamou E and Bougneres PF (1998) Ovarian function after autologous bone marrow transplantation in childhood: high-dose busulfan is a major cause of ovarian failure. Bone Marrow Transplant 22,989–994.[CrossRef][ISI][Medline]

Toft E, Becedas L, Soderstrom M, Lundqvist A and Depierre JW (1997) Glutathione transferase isoenzyme patterns in the rat ovary. Chem-Biol Interact 108,79–93.[CrossRef][Medline]

Volker P, Grundker C, Schmidt O, Schulz KD and Emons G (2002) Expression of receptors for luteinizing hormone-releasing hormone in human ovarian and endometrial cancers: frequency, autoregulation, and correlation with direct antiproliferative activity of luteinizing hormone-releasing hormone analogues. Am J Obstet Gynecol 186,171–179.[CrossRef][ISI][Medline]

Waxman JH, Ahmed R, Smith D, Wrigley PF, Gregory W, Shalet S, Crowther D, Rees LH, Besser GM, Malpas JS et al. (1987) Failure to preserve fertility in patients with Hodgkin’s disease. Cancer Chemother Pharmacol 19,159–162.[ISI][Medline]

Whitehead E, Shalet SM, Blackledge G, Todd I, Crowther D and Beardwell C (1983) The effect of combination chemotherapy on ovarian function in women treated for Hodgkin’s disease. Cancer 52,988–993.[ISI][Medline]

Zheng W, Magid MS, Kramer EE and Chen YT (1996) Follicle-stimulating hormone receptor is expressed in human ovarian surface epithelium and fallopian tube. Am J Pathol 148,47–53.[Abstract]





This Article
Extract
Full Text (PDF )
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Request Permissions
Google Scholar
Articles by Oktay, K.
Articles by Oktem, O.
PubMed
PubMed Citation
Articles by Oktay, K.
Articles by Oktem, O.