1 Institute of Reproductive Medicine, Salt Lake, Kolkata 700091, 2 School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302 and 3 Reproductive Biology Research, Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, West Bengal, India
4 To whom correspondence should be addressed at: Indian Institute of Chemical Biology, Jadavpur, Kolkata 700032, West Bengal, India.or; Email: syednkabir{at}yahoo.com
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Abstract |
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Key words: aromatase inhibitor/letrozole/low-cost IVF/poor ovarian responders
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Introduction |
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Aromatase inhibition has recently been focused on as an effective means of ovulation induction in the management of infertility (Mitwally and Casper, 2002a; b
; 2003a
; b
). Letrozole, a highly selective, non-steroidal aromatase inhibitor, could successfully induce ovulation in women with polycystic ovary syndrome (PCOS) (Mitwally and Casper, 2000
), including even those who were resistant to clomiphene citrate (Mitwally and Casper, 2001
). Recent studies demonstrated that addition of letrozole improved ovarian response to FSH in poor responders and reduced gonadotrophin dose required for COH in women with unexplained infertility (Mitwally and Casper, 2002a
; 2003a
). Healey et al. (2003)
demonstrated that addition of letrozole to gonadotrophins increased the number of pre-ovulatory follicles without having a negative impact on pregnancy rates. These reports prompted us to hypothesize that adjunctive use of letrozole in COH protocol would minimize the gonadotrophin dose, and consequently the cost, of an IVF treatment cycle. The present investigation is an endeavour to evaluate the potential of letrozolegonadotrophin combination therapy as a low-cost stimulation protocol in older women with poor ovarian response.
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Materials and methods |
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The Let-FSH group of patients received letrozole (Letroz; Sun Pharmaceuticals, Mumbai, India) at a dose of 2.5 mg daily orally from day 3 to 7 of the menstrual cycle, and rFSH (Gonal-F; Serono, Aubonne, Switzerland) was administered subcutaneously at a dose of 75 IU/day on days 3 and 8 of the menstrual cycle. The patients in the GnRH-ag-FSH group received daily subcutaneous injection of 500 µg of a GnRH agonist, leuprolide acetate (Lupride4; Sun Pharmaceuticals) starting from the mid-luteal phase of the previous cycle and continuing for a period of 14 days, or until the onset of the next menstruation, whichever was earlier. If the patient did not start menstruating by day 14 of GnRH agonist treatment, estradiol (E2) and LH levels were assayed and analysed. E2 levels 10 pg/ml or LH
3 mIU/ml were considered the presumptive evidences of down-regulation. However, if the patient did not meet the above criteria, GnRH agonist was continued for a further period of 4 days at the same dose level. The patients not reaching the set criteria for down-regulation even after extended GnRH agonist therapy were excluded from the study. rFSH was administered subcutaneously to the down-regulated subjects at a dose of 300 IU/day, with subsequent adjustment of the dose according to the doseresponse scheme.
All patients were monitored for ovarian follicular development by transvaginal ultrasonography. When the average diameter of the leading follicle(s) reached 18 mm, blood was drawn for the assessment of terminal E2 and they were administered 10 000 IU HCG (Profasi, Serono, Switzerland) subcutaneously as a single dose. Oocytes were retrieved by transvaginal ultrasonography
3436 h after HCG administration. The retrieved oocytes were inseminated with spermatozoa of the husband. Embryo transfer was performed 4042 h following insemination at 46 cell cleavage stages. All patients received 600 mg micronized progesterone (Utrogestan; Laboratories Besins International, Paris, France) intravaginally daily until a pregnancy test was performed, and if the test was positive, progesterone treatment was continued up to 12 gestational weeks. Clinical pregnancy was defined when an ultrasound scan, performed 5 weeks after embryo transfer, revealed the presence of a viable fetus.
Immunoassay of hormones
Serum levels of LH and FSH were measured by a two-site chemiluminescent sandwich immunoassay system (ACS:180; Bayer Diagnostics Corporation, Tarrytown, NY, USA). All samples were assayed in duplicate. The LH and FSH values were expressed in terms of the reference standards (WHO 2nd IS 94/632 and WHO 2nd IS 80/552, respectively). Assay sensitivity for FSH was 0.3 mIU/ml and for LH was 0.07 mIU/ml. E2 levels were assayed by fully automated enzyme-linked fluorescence assay system (Vidas; bioMerieux, Marcy l'Étoile, France). The minimum detection limit was 9 pg/ml. The intra- and inter-assay coefficients of variation were 3.46% and 4.82% for FSH, 4.4% and 5.6% for LH and 4.2% and 5.2% for E2, respectively.
The primary outcome measure was comparative evaluation of pregnancy outcome, while additional measures included total dose of FSH administered, the number of mature follicles, the levels of terminal E2, number of oocytes retrieved, endometrial thickness and transferable embryos.
Statistics
Statistical comparisons were performed using Student's t-test and 2-test, as applicable. P<0.05 was considered to be statistically significant.
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Results |
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Discussion |
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There are no consistent criteria to define poor responders. The definition is usually based on previous ovarian stimulation cycles, but the parameters vary between studies. In the present investigation, women over 35 years of age exhibiting emergence of fewer than two dominant follicles in response to conventional stimulation protocol were defined as elderly and poor responders. There are reports that the poor responders respond better to flare-up protocols than to standard long luteal protocols (Scott and Navot, 1994; Toth et al., 1996
). In our set-up, however, long protocol down-regulation is the method of choice, and irrespective of age and follicular reserve of the women, our experience with flare-up protocol is not encouraging. The result of the study group was therefore compared with those of the conventional long-term down-regulated protocol.
It is of particular importance to note that despite the use of 20-fold lower dose of FSH in the Let-FSH group, which entailed significant reduction of the cost of treatment, the outcomes in all major respects including pregnancy rate were comparable between the groups.
The precise mechanism of the ovarian effects of letrozole is as yet unexplored; however, some of the earlier observations and propositions on the effects of letrozole can be extrapolated to formulate a hypothesis. During the reproductive years, estrogens are chiefly produced in the ovary under the stimulation of aromatase. As the menopausal stage approaches, there occurs a decline in ovarian estrogen production; however, the extragonadal sites, notably adipocytes, continue to contribute peripheral production of estrogens that may act locally as paracrine or even intracrine factors (Labrie et al., 1997; Simpson et al., 2000
). Because of selective inhibition of aromatase, letrozole significantly inhibited the overall production of estrogens, which was reflected in the decreased levels of terminal E2 in the Let-FSH group. Consequent withdrawal of the negative feedback effects of estrogens may allow the pituitary to produce more endogenous FSH. Moreover, attenuated aromatization may secondarily lead to accumulation of follicular androgens, which may increase the follicular sensitivity through amplification of FSH receptor gene expression (Vendola et al., 1999
; Weil et al., 1999
) or stimulate insulin-like growth factor-I, which may act in synergy with FSH (Giudice, 1992
; Palter et al., 2001
). All these effects may have phenomenal importance in the letrozole-mediated promotion of follicular maturation. It may be significant in this context to emphasize that the absence of GnRH down-regulation in this proposed low-cost protocol may entail premature LH surge and luteinization, leading to cancellation of the index cycle. However, possibly due to the small sample size, this problem was not encountered in the present study.
This preliminary study, designed to evaluate the efficacy of Let-FSH as a low-cost IVF protocol, involved a small number of patients; however, the results have been encouraging. We have been successful in reaching our objective to modify an expensive conventional down-regulated IVF protocol into one in which the cost was reduced to a larger extent, without compromising the rate of success. A number of protocols of ovarian stimulation have been proposed for poorly responding women, but these have little or no proven benefit (Karande et al., 1990; van-Hooff et al., 1993
; Land et al., 1996
). Moreover, the cost of treatment, chiefly owing to the high cost of gonadotropins, is frequently prohibitive. Suggestions have therefore been made that natural cycle IVF, which may produce high-quality embryo(s) without a high cost involvement, may be considered for so-called elderly poor responders; however, likelihood of pregnancy has been reported to be low (Bar-Hava et al., 2000
). The present study bears the promise that as an alternative to natural cycle IVF, letrozole may have future prospects as a cost-saving stimulation protocol for IVF in women with poor ovarian response. Nevertheless, larger randomized studies are needed to confirm these data. It must also be taken into consideration that the use of letrozole as a low-cost IVF protocol, though exciting, has to be evaluated further carefully, as letrozole and other aromatase inhibitors have not been extensively used in women of reproductive age. This has been the main reason for employing selectively elderly women for this pilot study.
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Acknowledgements |
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References |
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Submitted on January 19, 2004; accepted on May 18, 2004.