1 Samuel Lunenfeld Research Institute and Mount Sinai Hospital, Reproductive Sciences Division, Department of Obstetrics & Gynecology, University of Toronto, Toronto, Canada and 2 Department of Gynecology and Obstetrics, State University of New York (SUNY) at Buffalo, Buffalo, New York, USA
3 Current address: Department of Gynecology and Obstetrics, State University of New York (SUNY) at Buffalo, 193 Kaymar Drive, Amherst, NY, 14228, USA
4 To whom correspondence should be addressed at: Samuel Lunenfeld Research Institute, Room 876, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5, Canada. e-mail: RFCasper{at}aol.com
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Abstract |
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Key words: aromatase inhibitor/clomiphene citrate/controlled ovarian stimulation/letrozole/unexplained infertility
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Introduction |
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A systematic review of randomized studies to evaluate the effectiveness of IUI demonstrated that pregnancy rates were significantly higher in women who received gonadotrophins, either FSH or hMG, compared with those who did not undergo COS (Hughes, 1997) prior to IUI. The reported pregnancy rates per cycle have usually varied between 8 and 22% (Sunde et al., 1988
; Dodson and Haney, 1991
; Peterson et al., 1994
; Brzechffa et al., 1998
; Cohlen et al., 1998
).
The rationale for COS in women with unexplained infertility, who by definition have regular ovulatory menstrual cycles, is to enhance the likelihood of pregnancy by increasing the number of oocytes available for fertilization and to overcome a possible subtle defect in ovulatory function not uncovered by conventional testing (Fisch et al., 1989). IUI, by increasing the density of motile sperm available to these oocytes, might further increase the monthly probability of pregnancy (Guzick et al., 1998
).
Clomiphene citrate (CC) has been used in the treatment of anovulatory infertility since 1962. By depleting the estrogen receptors, CC acts as an anti-estrogen on the central nervous system. This increases the pulse frequency of FSH and LH, giving a moderate gonadotrophin stimulus to the ovary and, thus, overcoming ovulatory disturbances and increasing the number of follicles reaching ovulation (Adashi, 1984; Dickey and Holtkamp, 1996
; Kousta et al., 1997
). Recently, sequential CC and gonadotrophin (hMG or FSH) therapy has become an increasingly utilized method of COS for patients who fail CC treatment (Kemmann and Jones, 1983
; Rose, 1992
; Dickey et al., 1993b
; Lu et al., 1996
). The value of adding CC during COS is to decrease the FSH dose required for optimum stimulation. However, CC use is associated with lower pregnancy rates because of its peripheral antiestrogenic effects offsetting the FSH dose reduction benefit.
Aromatase is a cytochrome P-450 haemoprotein-containing enzyme complex that catalyses the rate-limiting step in the production of estrogens, i.e. the conversion of androstenedione and testosterone into estrogens (Cole and Robinson, 1990; Akhtar et al., 1993
). The aromatase enzyme is a good target for selective inhibition because estrogen production is a terminal step in the biosynthetic sequence.
Recently, a group of highly selective aromatase inhibitors (AI), including letrozole and anastrozole, has been approved for use in post-menopausal women with breast cancer to suppress estrogen production. These AI have a relatively short half-life (48 h) compared with CC, and therefore would be eliminated from the body rapidly (Sioufi et al. 1997a
;b). In addition, since no estrogen receptor down-regulation occurs, no adverse effects on estrogen target tissues, as observed in CC-treated cycles, would be expected.
We hypothesized that it may be possible to mimic the action of CC, without depletion of estrogen receptors, by administration of an AI in the early part of the menstrual cycle. This use of an AI would result in release of the hypothalamicpituitary axis from estrogenic negative feedback, thereby increasing gonadotrophin secretion and resulting in stimulation of ovarian follicles.
In prior reports, we showed that aromatase inhibition is successful in inducing and augmenting ovulation without anti-estrogenic effects (Mitwally and Casper, 2000a;b;c; 2001). The objective of this study was to test the hypothesis that the use of the aromatase inhibitor, letrozole, in conjunction with FSH for COS, would decrease the dose of gonadotrophins required for COS similar to CC with FSH when compared with FSH only as a control.
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Materials and methods |
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This was a non-randomized prospective study, which included 110 ovulatory women with unexplained infertility or mild male factor infertility undergoing COS and IUI. None of the patients had polycystic ovarian syndrome or oligo-anovulation. Unexplained infertility was diagnosed by exclusion of known factors of infertility. Ovulation was confirmed with follicular monitoring by transvaginal sonography (TVS) and serial measurements of serum estradiol (E2) and LH hormone levels during a natural (no treatment cycle) and/or mid-luteal progesterone >15 nmol/l associated with regular menstrual cycle. Tubal patency was confirmed by hysterosalpingography and/or pelvic laparoscopy and male factor infertility was excluded by semen parameters meeting the World Health Organization (1999) criteria. All the study couples had
1 year of infertility, and had undergone at least one to three cycles of follicular monitoring with timed intercourse before undergoing COS and IUI with partners sperm.
The two standard protocols which are usually applied for COS in our study centres included the use of FSH either alone or in conjunction with CC. The addition of CC and the choice of the type and dose of FSH were usually decided according to the preference of the primary treating physician at the units. The treatment protocol was decided during a consultation visit prior to starting the treatment cycle. The choice was based on the clinical profile of the patient including age, weight, and duration of infertility as well as prior response to FSH and or CC.
Patients were counselled regarding the novel use of aromatase inhibitors to enhance ovarian response to FSH stimulation during COS. The experimental nature of the use of an aromatase inhibitor for ovulation induction was discussed. Thirty-six patients volunteered to use the aromatase inhibitor after the preliminary nature of the study was described to them and were included in the first study group. They received the aromatase inhibitor, letrozole (Femara®; Novartis, USA), 2.5 mg/day from day 3 to day 7 of the menstrual cycle, plus FSH injection [50150 IU/day starting on day 7 until the day of hCG (10 000 IU)]. Eighteen women in the second study group received CC (Serophene®; Serono, Canada) 100 mg from day 5 to day 9 of the menstrual cycle plus FSH injection [50150 IU/day starting on day 5 until the day of hCG (10 000 IU)]. FSH injection only [50225 IU/day starting on day 3 until the day of hCG (10 000 IU)] was given to 56 women constituting the control group for the first two study groups. Each patient received one treatment regimen in one treatment cycle only. All patients received recombinant FSH (Puregon®; Organon, Canada; or Gonal-F®; Serono) or highly purified FSH (Fertinorm®; Serono).
Because of the experimental nature of the use of an aromatase inhibitor for augmentation of ovulation, the patients were not randomized for this preliminary clinical trial and the choice of receiving an aromatase inhibitor was exclusively left to the patient. However, at the end of the study period, analysis of the patients characteristics revealed no significant difference among the two study groups and the control group in age, duration of infertility, or number of prior IUI cycles, as shown in Table I.
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IUI was performed 36 h after hCG administration if no endogenous LH surge occurred. If an endogenous LH surge occurred on the day of hCG administration, IUI was done on the following day. An LH surge was defined as an increase in LH level >100% over the mean of the preceding 2 days. IUI was performed by the same two infertility nurses for all patients.
Pregnancy was diagnosed by quantitative hCG 2 weeks after the insemination. Clinical pregnancy was confirmed by observing fetal cardiac pulsation 4 weeks after positive pregnancy test by TVS.
Statistical analysis
The various outcome measures are expressed as mean ± SD. The following statistical tests were used where appropriate to analyse the various data among the three groups (two study groups and one control group). Analysis of variance, group t-test or Students t-test, 2-test and Bonferroni t-test were used to compare data. P < 0.05 was considered statistically significant. The statistical tests were performed with SigmaStat for Windows Version 1.0 software (SigmaStat Software HighEdit Professional Copyright® 1993, MicroHelp Inc. and HeilerSoftware GmbH, USA).
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Results |
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Discussion |
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The reduced FSH dose required for COS associated with aromatase inhibition may be due to a central and/or a peripheral mechanism of action. Centrally, inhibition of estrogen synthesis by an aromatase inhibitor may release the estrogenic negative feedback on the hypothalamus and/or pituitary resulting in an increase in endogenous gonadotrophin secretion leading to enhancement of ovarian follicular development. Peripherally, at the ovarian level, inhibition of the conversion of androgens into estrogens by aromatase inhibition may lead to temporary accumulation of the androgens. Androgens were found to increase follicular sensitivity to FSH through amplification of the FSH receptor gene expression either directly or through other mediators such as the insulin-like growth factor system (Giudice, 1992; Adashi, 1993
; Vendola et al., 1998
; 1999; Weil et al., 1999
). Other mechanisms yet to be determined may also be working at the peripheral level and need further study to improve our understanding of follicular development in both health and disease.
Co-treatment with CC during COS has been proposed as a means to reduce the cost of gonadotrophin treatment (Dickey et al., 1993b). However, there is a well-known discrepancy between the high ovulation rates after CC treatment and a relatively low pregnancy rate. The lower pregnancy rates may be associated with anti-estrogenic effects of CC on the endometrium (Gonen and Casper, 1990
; Yeko et al., 1992
; Bonhoff et al., 1993
; Massai et al., 1993
; Wolman et al., 1994
; Hosie and Murphy, 1995
); and on the cervical mucus (Acharya et al., 1993
; Asaad et al., 1993
; Gelety and Buyalos, 1993
; Massai et al., 1993
). A decrease in uterine blood flow (Hsu et al., 1995
) and impairment of placental protein 14 synthesis (Johnson et al., 1993
) in addition to the increased subclinical pregnancy loss (Shoham et al., 1990
; Bateman et al., 1992
; Saunders et al., 1992
) have also been suggested as contributing to lower pregnancy rates with CC treatment. Moreover, detrimental effects of CC on tubal transport (Whitelaw et al., 1970
), and on the oocyte (Wramsby et al., 1987
) and embryo (Schmidt et al., 1985
; 1996; Laufer et al., 1983
; London et al., 2000
) have been suggested.
Whatever the mechanism(s) behind the lower pregnancy rate with CC treatment, the accumulation of CC in the body due to its long half-life (a few weeks) (Mikkelson et al., 1986) allows the occurrence of these deleterious effects. Due to the much shorter half-life, in addition to the absence of any antiestrogenic effects, we believe that use of the new aromatase inhibitors (third generation) would be associated with no deleterious effects on the final stages of follicular development and oocyte maturation or the early developing embryo. With a half-life of
45 h (Sioufi et al., 1997a
;b), administration of one of these new aromatase inhibitors in the early follicular phase should result in drug levels in the body that are extremely low or absent during the peri-ovulatory and luteal phases of the cycle.
Because measuring the endometrial thickness is relatively simple, non-invasive and routinely applied during follicular monitoring, we considered it as a clinically applicable method to monitor the antiestrogenic effects on the endometrium. Dickey and Holtkamp (1996) reported that in patients stimulated with CC for IUI, no pregnancy was observed when the endometrial thickness was 6 mm on the day of hCG administration, while all preclinical abortions occurred when endometrial thickness was 68 mm. In our present study, the endometrial thickness during CC treatment was significantly lower than the FSH-only and the letrozole + FSH groups. This finding is consistent with the persistent antiestrogenic effect of CC on endometrial thickness despite the high E2 levels associated with multiple follicular development. To overcome the anti-estrogenic effects of CC, various methods have been suggested, generally without success. These include starting CC early on day 2 or 3 of the cycle (Dickey and Holtkamp, 1996
; Triwitayakorn et al., 2002
), adding ethinyl estradiol in the follicular phase (Yagel et al., 1992
) and delaying administration of hCG (Dickey et al., 1993a
). The administration of other anti-estrogenic drugs was also tried without significant benefits (Boostanfar et al., 2001
).
Although the pregnancy rate in the CC + FSH group was statistically significantly lower when compared with the other two groups, we believe it is difficult to draw definitive conclusions from the present data regarding pregnancy rates. It is risky to compare outcomes in patients who were not randomized to the various treatment regimens. However, in the absence of any statistically significant difference between the patient groups in characteristics that might affect the achievement of pregnancy, we conclude that the pregnancy rate with aromatase inhibition is at the least acceptable while achieving the advantage of reducing the FSH dose needed for COS.
When the treatment cycles in which an endogenous LH surge occurred on the day of hCG administration were analysed separately (Tables VVII), endometrial thickness in the CC + FSH group was found to be significantly better. Moreover, the only two pregnancies achieved during CC + FSH treatment occurred in cycles with an endogenous LH surge. The occurrence of an endogenous LH surge might indicate release of the hypothalamus and/or pituitary from the anti-estrogenic effect of CC allowing rising estrogen to exert its positive feedback and triggering the LH surge. This might also explain the favourable treatment outcome in terms of achieving pregnancy. Unfortunately, during CC treatment it is not possible to predict which patient would clear CC fast enough to allow the hypothalamus, pituitary and peripheral genital tissues to escape the antiestrogenic effect of CC. In a recent study, we found a better outcome in terms of achievement of pregnancy associated with the occurrence of endogenous LH surge compared with when hCG was given to trigger ovulation in the absence of an LH surge. This favourable outcome was most significantly seen with CC treatment (Mitwally et al., 2002).
In analysing the cycles with an endogenous LH surge (Tables VVII), the mean LH level on the day of hCG administration was statistically significantly higher in the letrozole + FSH group when compared with the other two groups. Moreover, we found that the endogenous LH surge occurred significantly earlier in the FSH only group when compared with the letrozole + FSH group. We believe that the higher LH levels attained with letrozole treatment may indicate a more physiological LH surge due to the faster clearance of letrozole and the more physiological estrogen levels. The earlier occurrence of the LH surge during FSH only when compared with letrozole + FSH may reflect a premature LH surge due to the supraphysiological estrogen levels obtained during FSH stimulation.
The day of hCG administration was found to be later in the CC + FSH group. This might be due to the fact that, in CC + FSH treatment, patients started to receive treatment on day 5 of the menstrual cycle while in the other two treatment groups, treatment was started 2 days earlier (on day 3 of the menstrual cycle).
Gonadotrophins are used alone or in combination with CC to stimulate the growth and maturation of multiple oocytes. However, there is evidence from sharing of oocytes between a donor undergoing COS and a non-stimulated recipient that COS has a negative impact on implantation, independent of oocyte quality (Check et al., 1995). COS has also been found to be associated with unfavourable obstetric outcome (Tanbo, 1995
; Maman et al., 1998
). It is possible that supraphysio logical levels of estrogen, attained during ovarian stimulation, may explain the adverse effects of ovarian stimulation on the outcome of infertility treatment (Paulson et al., 1990b
; Hadi et al., 1994
; Simón et al., 1995
). Although the actual mechanism(s) of a possible adverse effect of high levels of estrogen on reproductive outcome are unknown, speculations include deleterious effects of estrogen on the endometrium (Garcia et al., 1984
; Forman et al., 1988
; Kolb et al., 1997
), the embryo (Pellicer et al., 1989
; Paulson et al., 1990a
; Ertzeid et al., 1992
; Warner et al., 1998
), the coagulation system (Kim et al., 1981
; Lox et al., 1995
), and the oviduct (Van der Auwera et al., 1999
).
One approach to improve treatment outcome has been to reduce the intensity of ovarian stimulation and subsequent estrogen levels. This approach includes minimal stimulation cycles or natural cycle IVF, and reducing the FSH dose (step-down protocol) to improve endometrial receptivity (Simón et al., 1998). However, these measures have the drawback of reducing the number of mature follicles, which is the main objective of ovarian stimulation. In our study, letrozole use was associated with significantly lower E2 levels when compared with the other stimulation protocols (FSH-only and CC + FSH), while resulting in the same number of mature follicles. Reducing estrogen synthesis by aromatase inhibition may therefore be a promising alternative to achieve a reduction in the serum concentration of estrogen in the peri-ovulatory and peri-implantation period, while still allowing multiple ovulation or retrieval of multiple oocytes in assisted reproductive treatment. Moreover, reducing the FSH dose needed for appropriate ovarian stimulation, as demonstrated in the present study, is an additional benefit. We have recently discussed the future avenues of applying aromatase inhibitors for infertility management (Mitwally and Casper, 2002b
) including the potential benefit for women with poor response to FSH ovarian stimulation as we reported in a preliminary series of poor responders (Mitwally and Casper, 2002a
).
A significant drawback in our present clinical trial is the non-randomized design of the study. However, due to the novel use of the aromatase inhibitors for ovarian stimulation, our institutional research board would not have approved a randomized study without a preliminary observational pilot trial. We believe that the present observational trial was mandatory to precede any definitive randomized studies in order to test for the feasibility of the idea of using aromatase inhibitors to reduce FSH dose needed for COS. We believe the positive results of the present trial should encourage us and others to proceed with more definitive prospective randomized clinical trials to prove or disprove our findings.
In summary, the results of the present study suggest that the concomitant use of the aromatase inhibitor, letrozole, during COS results in a reduction of the dose of FSH required to achieve a mean of three mature follicles prior to IUI without the deleterious peripheral anti-estrogenic effects often observed with CC. In addition, the low physiological levels of estrogen in the letrozole + FSH group may contribute to an improvement in pregnancy rates compared with the CC + FSH study group.
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References |
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Submitted on September 2, 2002; resubmitted on October 15, 2002; accepted on April 16, 2003.