1 Tecnobios Procreazione, Center for Reproductive Health, Via Dante 15, I-40125 Bologna, Italy and 2 University of Bologna, Bologna, Italy
3 To whom correspondence should be addressed. Email: borini{at}tecnobios.it
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Abstract |
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Key words: GnRH-agonist/half-dose/IVF/ovarian stimulation/triptorelin
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Introduction |
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Some authors have suggested that it might be enough to induce just partial pituitary desensitization in assisted reproductive techniques and have suggested protocols that use reduced doses of short-acting GnRH agonist (Feldberg et al., 1994; Olivennes et al., 1996
; Janssens et al., 2000
; Dal Prato et al., 2001
). In a previous study (Dal Prato et al., 2001
) we showed that reduced daily doses of short-acting triptorelin induce a lower level of pituitary suppression, which is nevertheless sufficient for ovarian stimulation in young women. However, although this treatment is shorter and requires a smaller amount of gonadotrophins, no significant improvement in IVF cycle outcome has been reported when compared with depot formulation.
Other studies have evaluated the effects of reducing depot formulation doses. Both full-dose (3.75 mg) and half-dose (1.87 mg) of GnRH agonist triptorelin seem to be equally effective in pituitary desensitization, with similar duration times for both desensitization and recovery (Balasch et al., 1992), but it remains to be determined which of the two doses provides the best clinical outcome in IVF treatment. Recently, a randomized trial (Yim et al., 2001
) showed that half-dose long-acting GnRH agonist is enough for the prevention of an LH surge in patients stimulated with hMG, but it does not improve IVF outcome in spite of the lower suppression. Since the presence of LH in the gonadotrophin formulation may compensate for low levels of endogenous LH during ovarian stimulation (Fleming et al., 1996
), it would be of interest to check the effect of half-dose long-acting GnRH agonist in patients stimulated with FSH only. The aim of this study was to compare the clinical efficacy of standard and half-doses of the depot GnRH agonist triptorelin, in long luteal protocol, prior to ovarian stimulation with highly purified FSH in IVF or ICSI.
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Materials and methods |
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Eligible patients who agreed to participate were randomized into two treatment groups.
Pituitary desensitization was obtained in group 1 (90 patients) with a half-dose (1.87 mg) triptorelin depot (Decapeptyl 3.75; Ipsen, Italy) in a single i.m. injection in mid-luteal phase (day 21) of the menstrual cycle preceding treatment, and in group 2 (90 patients) with standard full dose (3.75 mg) in the mid-luteal phase of the menstrual cycle preceding treatment. At the onset of menses, patients in both groups began gonadotrophin stimulation as described elsewhere (Dal Prato et al., 2001). Briefly, they received 4 ampoules (300 IU) per day of highly purified urofollitropin (Metrodin HP 75; Serono, Italy) for 2 days and 2 ampoules (150 IU) per day for 4 days. The dose was then adjusted according to the individual response as assessed by 17
-oestradiol (E2) assay and ultrasound scanning performed every other day. Metrodin HP was chosen, as a drug containing merely FSH, because it was the only option allowed by our local health authority for the first IVF attempt, without charging the whole cost of the medication to patients.
hCG (Profasi HP; Serono, Italy), 10 000 IU, was administered when at least three follicles reached a maximum diameter of 20 mm, of which at least one was >23 mm. E2 levels were only used as an indication for coasting, when they were >3000 pg/ml, for the prevention of ovarian hyperstimulation syndrome (OHSS). Cycles in which less than three follicles developed were discontinued. Transvaginal oocyte retrieval was performed under ultrasound guidance 36 h after hCG administration.
Two days after oocyte retrieval, a maximum of two embryos in women aged <35 years and three in women aged >35 years were replaced into the uterine cavity via the transcervical route. All remaining embryos presenting adequate morphology were cryopreserved for future use.
Luteal phase support was sustained with natural progesterone in oil (Prontogest; AMSA, Italy), 50 mg i.m. daily from day 1 after oocyte retrieval.
Pregnancy was defined as the presence of one or more gestational sacs detected on ultrasound scan performed 4 weeks after embryo transfer. Biochemical pregnancies (a rise of±hCG with no further evidence of a gestational sac on ultrasound scan) were not considered.
Parameters evaluated
The primary endpoint was the total number of oocytes retrieved from patients who received hCG. The following secondary endpoints were also recorded: number of FSH ampoules, number of days of stimulation, 17-oestradiol (E2) at hCG administration, LH on day 7 of stimulation and at hCG administration, progesterone at hCG administration, number of follicles at hCG administration, number of mature oocytes, fertilization rate, number of cleaved and grade 1 embryos, number of embryos transferred or frozen, number of clinical pregnancies, miscarriages and multiple pregnancies.
Sample size
Sample size calculations were based on 80% power and 5% significance level. Calculations were based on the following assumptions for number of oocytes: expected means difference 2.00 (Dal Prato et al., 2001). Under these conditions,
90 patients were required in each group of the study.
Assignment
Randomization was performed on an individual basis using sealed envelopes containing the name of one of the two medications (triptorelin 1.87 or triptorelin 3.75). Assignment to the different dosage groups occurred when eligible patients agreed to participate, 2 weeks before triptorelin administration. Dark envelopes were used so that their content could not be seen against bright light. Each envelope and allocation was sequentially numbered to avoid patients being randomized out of sequence. Envelopes were not allowed to be opened in advance and were opened only by a nurse not involved in the trial, but expressly charged with the drug injections. Both patients and physicians involved were blinded to the dosing.
Statistical analysis
Analysis was performed using an SPSS program on an intention-to-treat basis (all patients included in the study) and per protocol (available patients). The results of the two analyses were similar; therefore only the results of the analysis per protocol are presented in detail.
Comparisons of clinical outcomes with half-dose (1.87 mg i.m.) and full-dose (3.75 mg i.m.) depot triptorelin and quality of the embryos between groups were analysed by 2-test for dichotomous variables (Fisher's exact test when appropriate). Student's t-test (MannWhitney U-test when appropriate) and Z-test for proportions were used for continuous variables of ovarian stimulation and quality of the embryos. Mean±SEM and KolmogorovSmirnov test to describe distributions and to verify normality were also used. P<0.05 was considered statistically significant.
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Results |
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LH and progesterone assays performed in each patient on the day of hCG administration showed no evidence of premature LH surge or luteinization in either group. Table I shows the hormonal pattern of the two groups of patients during ovarian stimulation. LH levels were lower in the full-dose group (group 2): on day 7 of stimulation the difference was barely significant (1.08±0.06 IU/l in group 1 versus 0.9±0.06 in group 2: P=0.035), but became highly significant on the day of hCG administration (1.04±0.05 IU/l versus 0.75±0.03: P=0.0007).
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The characteristics of ovarian stimulation are summarized in Table II. The number of FSH ampoules used (42±2 versus 59±3: P=0.0001) and length of stimulation (11.8±0.13 versus 12.4±0.12: P=0.0143) were lower in group 1 which also presented significantly more follicles (total and with mean diameter >17 mm) at hCG administration. The number of collected oocytes and of mature oocytes, the fertilization rate and the number of cleaved embryos were higher in group 1. There was no difference in the number of embryos transferred, but there were more embryos suitable for cryopreservation in group 1. Evaluation of the quality of embryos according to embryo grading (Hu et al., 1998) showed a significantly higher rate of G1 embryos in the group treated with half-dose triptorelin (Table III).
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Discussion |
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Janssens et al. (2000) was the first to demonstrate a direct correlation between the reduction of the daily dosage of short-acting triptorelin and the decrease in the degree of pituitary suppression. Balasch et al. (1992)
reported that half-dose depot triptorelin is enough for preventing an LH surge. More recently Hsieh et al. (2000)
showed that a single half-dose of leuprolide acetate depot gives pituitary suppression and clinical results comparable with those of 0.5 mg daily leuprolide acetate.
In our study, the effect of the differently dosed drugs on the pituitary was quite different in the two groups, as can be seen from the levels of LH in mid- and late follicular phases, that confirm the reduced pituitary desensitization induced by half-dose triptorelin. Despite this difference between the mean LH levels of the two groups, 1.87 mg triptorelin administration proved sufficient to prevent an LH surge. LH and progesterone assay performed at hCG administration provided no evidence of an LH surge or premature luteinization in any of our patients. Moreover, oocytes were found at each retrieval and the number of post-mature or degenerative oocytes was very low.
Recently a randomized trial in which patients were stimulated with hMG (Yim et al., 2001) reported similar findings. Pituitary suppression was significantly lower with half than with the conventional dose, but this did not affect IVF outcome. There was no difference in the gonadotrophin doses used, the number of oocytes and embryos, or pregnancy rates.
In contrast, the difference in LH levels in our study seems to have affected the clinical outcome. In the patients treated with half-dose, quicker stimulation, requiring a smaller amount of gonadotrophins, led to a larger number of oocytes collected and, what is more, a larger number of oocytes being classified as mature via cumulusoocyte complex examination. The higher number of fertilized oocytes obtained in this group is probably due to the better quality of oocytes collected.
These findings contrast with the work by Janssens et al. (2000). He found a direct correlation between the GnRH agonist dose and the number of follicles and oocytes. On the contrary, in the present study, the lowest depot GnRH agonist dose gives rise to a higher number of follicles and oocytes. The difference between the two studies might result from the different drug formulation administered: depot in the present study, daily in Janssen et al.'s work. Moreover the levels of LH reported by Janssens et al. with very low daily GnRH agonist doses seem to be higher than those achieved in our trial. Tesarik and Mendoza (2002)
investigated the effect of exogenous LH in oocyte donors stimulated with rFSH and suggested the concept of a window for LH requirement in ovarian follicular development. In donors with profound pituitary suppression (LH levels <0.1 IU/l), the addition of exogenous LH increased the number of mature oocytes and good quality embryos. In contrast, when LH levels at the beginning of stimulation were >1 IU/l, exogenous LH administration impaired oocyte and embryo quality and decreased the implantation rate in recipients.
The actual quality of the embryos is an interesting issue. In group 1 there were not only more embryos available for cryopreservation, there was also a higher rate of grade 1 embryos. A larger number of best morphology embryos available for transfer or cryopreservation resulted in a higher implantation rate after both fresh and thawed transfer. Combining the results of the fresh and frozenthawed embryo transfers, the final outcome of the treatment cycle was therefore significantly better in the group treated with half-dose triptorelin.
The difference between the results of our study and those reported by Yim et al. (2001) lies probably in the kind of gonadotrophin used: hpFSH (virtually devoid of LH activity) in the present study, hMG (with LH activity) in that of Yim et al. The administration of exogenous LH in the latter may have partially balanced the effects of the different degrees of pituitary suppression on the quality of ovarian stimulation.
According to the two-cell two-gonadotrophin theory, both FSH and LH are required for a normal follicular (and oocyte) growth and maturation. However, the actual role of LH in ovarian stimulation is a matter of debate. Overly high LH levels during the follicular phase may induce excess follicular androgen secretion, thus increasing follicular atresia and producing low quality embryos (Loumaye et al., 1989). On the other hand, Fleming et al. (1996)
showed that follicular fluid E2 levels, oocyte yield and fertilization are decreased and the follicular phase is longer when LH concentrations are <1 IU/l, indicating a reduction in normal follicular steroid metabolism. These findings were confirmed in a subsequent report (Fleming et al., 1998
) in women with more profoundly suppressed mid-follicular phase LH (<0.5 IU/l), who had significantly fewer embryos available for cryopreservation. The rate of blastocyst formation, however, was normal, indicating that embryo development potential is not impaired by the degree of LH suppression.
Moreover, it has been recently suggested that a direct action of LH on uterine LH receptors is required to support endometrial growth and uterine receptivity (Tesarik, 2003), and that the expression of endometrial estrogens and progesterone receptors is altered in COS when GnRH agonist is used and in which LH levels are low (Bourgain et al., 2002
).
Filicori et al. (2001) compared highly purified FSH and hMG in normo-ovulatory GnRH agonist-suppressed women, candidates for intrauterine insemination, and found a shorter treatment duration with lower gonadotrophin consumption in the hMG-treated group, underlining a facilitatory role of LH activity in ovulation induction.
Thus, evidence in the literature suggests that there may be a role for LH co-administration in cycles treated with depot GnRH agonist and FSH. However, the good results achieved in our trial suggest that lowering the dose of depot GnRH agonist may reduce the LH requirement in patients undergoing assisted reproduction treatment. This management may be preferable to screening for women that might benefit from the addition of exogenous LH. This approach seems simpler and cheaper. In view of the high costs of assisted reproduction treatment procedures, shorter treatment with smaller amounts of drugs for stimulation might be a financial advantage for patients.
As a final consideration, specific GnRH receptors have been found in murine and human granulosa cells (Bramley et al., 1985; Latouche et al., 1989
) and GnRH agonist has been shown to have a direct effect on ovarian steroidogenesis (Casper and Yen, 1979
; Tureck et al., 1982
; Parinaud et al., 1988
). More recently Zanagnolo et al. (1996)
observed that pharmacological doses of leuprolide may exert a negative effect on oocyte function in the rabbit by direct action on the oocyte. These findings should furthermore suggest the use of the possibly lower dose of GnRH agonist.
In conclusion, the results of our study show that half-dose (1.87 mg) depot triptorelin can be successfully used in ovarian stimulation for IVF or ICSI. LH levels confirm the lower level of pituitary desensitization elicited by half-dose triptorelin. The better quality oocytes recovered in the half-dose group might explain the higher fertilization rate as well as the higher number of good quality embryos available for transfer or cryopreservation, and subsequently the higher cumulative pregnancy rate.
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Acknowledgements |
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Notes |
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References |
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Ben-Rafael Z, Lipitz S, Bider D and Mashiach S (1991) Ovarian hyporesponsiveness in combined gonadotropin-releasing hormone agonist and menotropin therapy is associated with low serum follicle-stimulating hormone levels. Fertil Steril 55, 272275.[ISI][Medline]
Bourgain C, Ubaldi F, Tavaniotou A, Smitz J, Van Steirteghem AC and Devroey P (2002) Endometrial hormone receptors and proliferation index in the periovulatory phase of stimulated embryo transfer cycles in comparison with natural cycles and relation to clinical pregnancy outcome. Fertil Steril 78, 237244.[ISI][Medline]
Bramley TA, Menzies GS and Baird DT (1985) Specific binding of gonadotrophin-releasing hormone and an agonist to human corpus luteum homogenates: characterization, properties, and luteal phase levels. J Clin Endocrinol Metab 61, 834841.[Abstract]
Casper RF and Yen SS (1979) Induction of luteolysis in the human with a long-acting analog of luteinizing hormone-releasing factor. Science 205, 408410.[ISI][Medline]
Caspi E, Ron-El R, Golan A, Nachum H, Herman A, Soffer Y and Weinraub Z (1989) Results of in vitro fertilization and embryo transfer by combined long-acting gonadotropin-releasing hormone analog D-Trp-6-luteinizing hormone-releasing hormone and gonadotropins. Fertil Steril 51, 9599.[ISI][Medline]
Dal Prato L, Borini A, Trevisi MR, Bonu MA, Sereni E and Flamigni C (2001) Effect of reduced dose of triptorelin at the start of ovarian stimulation on the outcome of IVF: a randomized study. Hum Reprod 16, 14091414.
Feldberg D, Farhi J, Ashkenazi J, Dicker D, Shalev J and Ben-Rafael Z (1994) Minidose gonadotropin-releasing hormone agonist is the treatment of choice in poor responders with high follicle-stimulating hormone levels. Fertil Steril 62, 343346.[ISI][Medline]
Filicori M, Cognigni GE, Taraborrelli S, Spettoli D, Ciampaglia W, Tabarelli De Fatis C, Pocognoli P, Cantelli B and Boschi S (2001) Luteinzing hormone activity in menotropins optimizes folliculogenesis and treatment in controlled ovarian stimulation. J Clin Endocrinol Metab 86, 337343.
Fleming R, Chung CC, Yates RW and Coutts JR (1996) Purified urinary follicle stimulating hormone induces different hormone profiles compared with menotrophins, dependent upon the route of administration and endogenous luteinizing hormone activity. Hum Reprod 11, 18541858.[Abstract]
Fleming R, Lloyd F, Herbert M, Fenwick J, Griffiths T and Murdoch A (1998) Effects of profound suppression of luteinizing hormone during ovarian stimulation on follicular activity, oocyte and embryo function in cycles stimulated with purified follicle stimulating hormone. Hum Reprod 13, 17881792.[Abstract]
Hsieh Y, Tsai H, Chang C and Lo H (2000) Comparison of a single half-dose, long-acting form of gonadotropin-releasing hormone analog (GnRH-a) and a short-acting form of GnRH-a for pituitary suppression in a controlled ovarian hyperstimulation program. Fertil Steril 73, 817820.[CrossRef][ISI][Medline]
Hu Y, Maxson WS, Hoffman DI, Ory SJ, Eager S, Dupre J and Lu C (1998) Maximizing pregnancy rates and limiting higher-order multiple conceptions by determining the optimal number of embryos to transfer based on quality. Fertil Steril 69, 650657.[CrossRef][ISI][Medline]
Janssens RM, Lambalk CB, Vermeiden JP, Schats R, Bernards JM, Rekers-Mombarg LT and Schoemaker J (2000) Dose-finding study of triptorelin acetate for prevention of a premature LH surge in IVF: a prospective, randomized, double-blind, placebo-controlled study. Hum Reprod 15, 23332340.
Latouche J, Crumeyrolle-Arias M, Jordan D, Kopp N, Augendre-Ferrante B, Cedard L and Haour F (1989) GnRH receptors in human granulosa cells: anatomical localization and characterization by autoradiographic study. Endocrinology 125, 17391741.[Abstract]
Liu H, Lai Y, Davis O, Berkeley A, Grifo M, Grifo J, Cohen J and Rosenwaks Z (1992) Improved pregnancy outcome with gonadotropin releasing hormone agonist stimulation is due to the improvement in oocyte quantity rather than quality. J Assist Reprod Genet 9, 338342.[ISI][Medline]
Loumaye E, Vankrieken L, Depreester S, Psalti I, de Cooman S and Thomas K (1989) Hormonal changes induced by short-term administration of gonadotropin-releasing hormone agonist during ovarian hyperstimulation for in vitro fertilization and their consequences for embryo development. Fertil Steril 51, 105111.[ISI][Medline]
Olivennes F, Righini C, Fanchin R, Torrisi C, Hazout A, Glissant M, Fernandez H and Frydman F (1996) A protocol using a low dose of gonadotrophin-releasing hormone agonist might be the best protocol for patients with high follicle-stimulating hormone concentrations on day 3. Hum Reprod 11, 11691172.[Abstract]
Parinaud J, Beaur A, Bourreau E, Vieitez G and Pontonnier G (1988) Effect of a luteinizing hormone-releasing hormone agonist (Buserelin) on steroidogenesis of cultured human preovulatory granulosa cells. Fertil Steril 50, 597602.[ISI][Medline]
Tesarik J (2003) Luteining hormone affects uterine receptivity independently of ovarian function. RBM Online 7, 5964.[Medline]
Tesarik J and Mendoza C (2002) Effects of exogenous LH administration during ovarian stimulation of pituitary down-regulated young oocyte donors on oocyte yield and developmental competence. Hum Reprod 17, 31293137.
Tureck RW, Mastroianni L, Jr, Blasco L and Strauss JFD (1982) Inhibition of human granulosa cell progesterone secretion by a gonadotropin-releasing hormone agonist. J Clin Endocrinol Metab 54, 10781080.[ISI][Medline]
Yim SF, Lok IH, Cheung LP, Briton-Jones CM, Chiu TT and Haines CJ (2001) Dose-finding study for the use of long-acting gonadotrophin-releasing hormone analogues prior to ovarian stimulation for IVF. Hum Reprod 16, 492494.
Zanagnolo V, Dharmarajan AM, Hesla J and Wallach EE (1996) Effects of a gonadotropin-releasing hormone analog on rabbit ovarian function. Endocrinology 137, 54005406.[Abstract]
Submitted on July 31, 2003; resubmitted on May 14, 2004; accepted on June 22, 2004.
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