1 IVI-Madrid, Madrid, 2 Instituto Valenciano de Infertilidad (IVI) and 3 Department of Paediatrics, Obstetrics and Gynaecology, Valencia University School of Medicine, Valencia, Spain
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Abstract |
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Key words: gonadotrophin/GnRH analogue/IVF/low responders
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Introduction |
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Low response to ovarian stimulation is often age-related, but this situation also occurs in young patients. Elevated basal or clomiphene-induced FSH concentrations may characterize the age-related decline in reproductive performance. However, some young patients with normal FSH concentrations present repeated low responses to aggressive stimulation protocols. In this particular group of patients, several hypothesis have been raised to explain the low ovarian response, but none has been proved. There is no doubt that whatever the underlying mechanism is, these patients have a diminished ovarian reserve, as has been recently demonstrated by three-dimensional ultrasound (Pellicer et al., 1998).
Clinicians have approached low responders in many different ways, using different dosages of gonadotrophin releasing hormone agonist (GnRHa), increasing the dose of gonadotrophins, co-treatment with oestrogens, growth hormone, clomiphene citrate or contraceptive pills, and even natural cycles (Scott, 1996; Karande and Gleicher, 1999
). The optimal approach for poor responders to ovarian stimulation is still controversial. Recently, a new protocol in which early cessation of GnRHa administration was combined with high doses of gonadotrophins yielded favourable results in low responder patients (Faber et al., 1998
). This retrospective report was followed by a prospective, randomized trial in which this approach offered no further advantage to these patients (Dirnfield et al., 1999
). Recent evidence confirms that early follicular GnRHa cessation is still effective in the prevention of a premature rise in LH (Valbuena et al., 1997
; Beckers et al., 2000
). The so-called stop protocol may improve ovarian responsiveness based on a hypothetical effect on the ovary, directly via GnRH receptor or indirectly regulating the vascular network within the ovary.
In a prospective, randomized clinical trial the hypothetical benefit of withholding GnRH administration upon down-regulation combined with high doses of gonadotrophins in women who previously had insufficient ovarian response to complete an IVF attempt was evaluated.
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Materials and methods |
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To be included in this study, patients had to have at least one previous cancelled IVF attempt in which fewer than three follicles 18 mm in diameter were obtained and basal FSH concentrations were <12 IU/ml (Karande and Gleicher, 1999
). There were no exclusion criteria, nor was there any age limit. The cancelled cycle was stimulated with a standard long protocol, as previously described (Simón et al., 1998
). Briefly, in our standard long protocol, after pituitary desensitization with leuprolide acetate, 1 mg/day s.c. (Procrin; Abbot S.A., Madrid, Spain) on days 1 and 2 of ovarian stimulation, one ampoule of human menopausal gonadotrophin (HMG; Lepori; Farma-Lepori Laboratories S.A., Madrid, Spain) was administered together with three ampoules of highly purified FSH (NeoFertinorm; Serono Laboratories S.A., Madrid, Spain). On days 3, 4 and 5 of ovarian stimulation, one ampoule of HMG and one ampoule of FSH were given to each patient. Beginning on day 6, HMG and FSH were administered on an individual basis according to serum oestradiol concentrations and transvaginal ovarian ultrasound scans.
During their next cycle, the patients were informed about the possibility of being included in the study with two study groups (stop protocol versus non-stop protocol). Thirty-six patients were treated in 36 cycles with a high dose regimen, whereas 34 were treated in 34 cycles with a stop protocol and a high dose regimen. Successive ovarian stimulation was separated by a minimum of two or more months in order to avoid any potential source of error, as this is our routine clinical practice.
Assignment and masking
Considering low ovarian response to ovarian stimulation as less than three oocytes retrieved, the primary endpoint was to at least duplicate the number of oocytes recovered. For that purpose a prospective study was designed in which 70 patients were included to be able to detect a true effect with this protocol with a probability of 80% (power), being the chances of incorrectly rejecting the null hypothesis 5% (alpha). Patients were randomized according to a computerized random number list to either intervention (stop protocol) or control group (non-stop protocol) at the time of previous cycle cancellation. Someone not involved in the study performed the randomization. The therapeutic choice was included in consecutively numbered opaque sealed envelopes.
Patients and stimulation protocol
The causes of infertility for patients who entered the study were as follows: in 26 cases (37.1%) there was male factor infertility, in eight cases (11.4%) tubal infertility, in 15 cases (21.4%) there was unexplained infertility, and in 21 cases (30%) a combination of male and female factors was present. The aetiology of infertility was equally distributed between groups. Mean age of the patients included in the high dose protocol and in the stop protocol was 34.0 ± 0.5 years and 34.4 ± 0.7 years respectively; body mass index of the patients included was 21.7 ± 2.5 and 21.9 ± 2.7 kg/m2 respectively.
The protocol for ovarian stimulation was initiated with pituitary desensitization by the administration of leuprolide acetate, 1 mg/day s.c. (Procrin; Abbot S.A., Madrid, Spain), starting in the luteal phase of the previous cycle, on cycle day 21, and this was adjusted according to the length of patient's cycle in order to start the analogue 7 days prior to menstruation. Serum oestradiol concentrations of <60 pg/ml (220 pmol/l) and negative findings, i.e. absence of ovarian cysts of >10 mm on vaginal ultrasound scans, were used to define ovarian quiescence. If a cyst >10 mm was observed, serum oestradiol concentrations <60 pg/ml was sufficient to confirm ovarian quiescence. If serum oestradiol concentrations were beyond this threshold value, the patient was excluded from the study.
For the high dose, non-stop protocol, a similar approach was used (long protocol) but on days 1 and 2 of ovarian stimulation, three ampoules of HMG were administered together with five ampoules of FSH. On days 3, 4 and 5 of ovarian stimulation, two ampoules of HMG and thre ampoules of FSH were administered. From day 6 onward, gonadotrophin dosage was estimated according to serum oestradiol concentrations and transvaginal ovarian ultrasound scans. Patients included in the stop protocol group received the same gonadotrophin doses as in the high dose, non-stop protocol group.
In both protocols, the criteria for HCG administration (10 000 IU, Profasi; Serono Laboratories) was the same. The criteria for HCG administration included the presence of at least three follicles measuring 18 mm in diameter. Gonadotrophin administration, and leuprolide acetate in non-stop protocol, were discontinued the day of HCG administration. Oocyte retrieval was scheduled for 3638 h after HCG injection and intravaginal micronized progesterone, 400 mg/day (Progeffik; Effik Laboratory, Madrid, Spain), was administered as luteal support.
IVF/intracytoplasmic sperm injection
The standard IVF/intracytoplasmic sperm injection (ICSI) procedure has been described previously (Pellicer et al., 1989; Gil-Salom et al., 1995
). Briefly, oocyte-cumulus complexes were evaluated under the dissecting microscope and classified. Oocyte-cumulus complexes were incubated at 37°C under 5% CO2 in atmospheric air. Embryos were scored on the day of transfer according to their morphology under the dissecting microscope. Four types of embryos were established, ranging from types I to IV. Type I embryos were the best and were defined as round and well-shaped blastomeres without fragments. The policy for embryo transfer was to select as many type I and type II embryos as possible: the remaining embryos were cryopreserved with 1,2-propanediol and sucrose. Only patients with freshly transferred embryos were included in the study. The maximum number of embryos transferred was three or four, depending on previous IVF attempts and age of the patient.
Hormone measurements
Blood was drawn with every transvaginal ovarian ultrasound scan. The samples were stored at 20°C in aliquots for subsequent oestradiol analysis. Serum oestradiol and FSH were analysed using commercially available microparticle enzyme immunoassay kits (Abbot Laboratories, Abbot Park, IL, USA). Inter- and intra-assay variability for oestradiol at a concentration of <40 pg/ml was 2.8 and 4.3%, respectively, and 4.6 and 6.1% for FSH at a concentration of 22 IU/ml.
Statistical analysis
Data were expressed as means ± SEM. Comparisons between different cycles of the same patient were performed with the use of the Student's t-test, except that pregnancy and implantation rates were compared with the use of 2-test. When homogeneity and normality (Kolmogorov-Smirnov test) of the samples were not appropriate, non-parametric statistical methods (Wilcoxon test) were used for comparison. P < 0.05 was considered significant. Statistical calculations were performed using Sigmastat for Windows, version 2.0 (Jandel Scientific Corporation, San Rafael, CA, USA).
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Results |
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Table II shows that in patients undergoing ovarian stimulation with the stop protocol a significantly higher number of oocytes was retrieved (8.7 versus 6.2, P = 0.027) as well as a higher number of mature oocytes (6.8 versus 4.1, P = 0.018). However, similar fertilization and cleavage rates were obtained. Also, the mean number of embryos transferred (2.8 versus 2.6) was similar. No differences were found in cancellation rate (5.8 versus 2.7%), pregnancy rate (18.7 versus 14.3%) and implantation rate between both treatment groups (12.1 versus 8.8%) (Table II
).
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Obviously, a higher number of FSH/HMG ampoules were administered in the high dose protocol (irrespective of whether the patient was included in the stop or non-stop protocol) when compared to the previous cancelled cycle (62.3 versus 35.5, P < 0.001). For the same reason, the number of days of stimulation (10.7 versus 9.5) as well as the peak oestradiol concentration (783.5 versus 367.2 pg/ml) was also higher in this group.
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Discussion |
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Several mechanisms may contribute to the improved ovarian response observed with the stop protocol. The pituitary gonadotrophin down-regulation induced by GnRHa decreases cancellation rates by suppressing endogenous LH surges, although this may require significantly higher requirements of gonadotrophins (Horvarth et al., 1988), probably due to a direct, partial inhibition of ovarian response, modulating ovarian steroidogenesis and oocyte maturation. A direct inhibitory effect of the GnRHa on the ovaries has been proposed, so reducing the dose or even stopping the administration would remove this suppression, improving ovarian responsiveness (Kowalik et al., 1998). This hypothesis is based on the presence of GnRH receptors on the ovary (Latouche et al., 1989
). On the other hand, GnRHa have been proved to decrease blood flow assessed by pulsed Doppler analysis (Aleem and Predanic, 1995
). Some investigators believe that follicular growth is dependent on an appropriate vascular network responsible for the distribution of circulating gonadotrophins (Zeleznik et al., 1981
). In fact, ovarian blood flow velocity, after pituitary suppression is confirmed, has been shown to be predictive of ovarian responsiveness and the outcome of IVF treatment (Engmann et al., 1999
). Thus, it could be postulated that GnRHa early cessation, while maintaining pituitary suppression, restores the diminished perifollicular blood flow, which correlates with the number of oocytes retrieved and IVF outcome (Bhal et al., 1999
). Whether this is true or not for ovarian vessels in low responders remains to be elucidated.
GnRHa is routinely used until oocyte retrieval, but even with short-acting molecules pituitary down-regulation continues following cessation of GnRHa during ovarian stimulation for IVF (Sungurtekin and Jansen, 1995). In a recent prospective study, the follicular and luteal phase characteristics following early cessation of GnRHa during ovarian stimulation were evaluated (Beckers et al., 2000
). Early follicular phase cessation of GnRHa is still effective in the prevention of a premature rise in LH or progesterone, as some pituitary recovery occurred 1622 days after GnRHa cessation. This is in accordance with the fact that most of the cancellations in the study group in the current study as well as in others (Faber et al., 1998
; Dirnfield et al., 1999
; Karande and Gleicher, 1999
) were not due to a premature ovulation.
When the previous cancelled cycles with a conventional IVF protocol were compared using the study cycles (either high dose or high dose combined with early cessation of GnRHa) a significant improvement in the ovarian response was found. It is interesting to note that regardless of the protocol used, stop or non-stop, we found that a satisfactory number of oocytes was retrieved in both groups of re-stimulated patients. If it is considered that they were enrolled in this study on the basis of a previous cancelled stimulation with less than three mature follicles, the only explainable hypothesis is the use of high doses of gonadotrophins. Although this explanation appears reasonable, conflicting reports have been published. Increasing the gonadotrophin dose during the course of treatment was initially advocated by Laufer (Laufer et al., 1986), in an attempt to recruit a higher number of follicles. Several other reports including our data support this approach (Chong et al., 1986
; Crosignani et al., 1989
). However, other authors have not reported such a beneficial effect (Karande and Gleicher, 1990; Van-Hoof et al., 1993
; Land et al., 1996
). Patients show a great variability in their ovarian response to the same stimulation protocol, especially those who qualify as poor responders to IVF stimulation, probably based on the intercycle variability of basal FSH concentrations (Scott et al., 1990
). Basal day 3 FSH concentrations were comparable between groups, so it is believed that the higher dose administered during the study cycles allowed the recruitment of a higher number of follicles and enabled most women to finish their cycle and undergo oocyte retrieval. Whether the FSH threshold in these women is higher or if the intra-individual variability in the ovarian response is responsible for the outcome is something to be determined.
The primary efficacy result, i.e. a significantly higher number of mature oocytes, was supported by the fact that the number of days of treatment and the dose of FSH required to reach criteria for triggering follicle maturation were both lower for the stop protocol. Gonadotrophin ampoule consumption was lower in the stop protocol, and less GnRHa was used, so the cost of this cycle in terms of medication is significantly reduced. The reduced gonadotrophin usage together with the higher number of mature oocytes retrieved make this protocol appealing, something that would be definitive if it allowed higher pregnancy rates. Although a trend was observed (18.7 versus 14.3%), a prospective study with pregnancy rate as primary end-goal should be designed to prove this hypothesis. The stop protocol, in spite of having significantly more oocytes (8.7 ± 0.9 versus 6.2 ± 0.7), had similar oestradiol concentrations to those observed in the non-stop protocol. In fact, serum concentrations of oestradiol were higher in the stop protocol, although the values did not reach statistically significant differences (859.5 ± 132.7 versus 708.6 ± 107.2 pg/ml, P = 0.380), probably due to the limited sample size of this study.
The `flare effect' derived by the GnRHa administration during the luteal phase does not seem to be relevant in the ovarian stimulation outcome in this kind of patient. San Roman (San Roman et al., 1992) did not find a significant increase in gonadotrophin concentrations in patients receiving GnRHa initiated in the luteal phase opposed to the concentrations found in patients who were initiated with GnRHa during the follicular phase. Consistent findings were described by Gelety (Gelety et al., 1995
).
When clinicians observe a low response to IVF stimulation (less than three follicles), cycle cancellation is usually counselled to the patients, in the hope that a better response might be obtained in a subsequent cycle. This trend is based on the assumption that poor responders have a poor IVF outcome, as they usually have a low oocyte quality. Not all low responders are the same, as they represent a very heterogeneous group. According to their basal FSH concentrations, young low responders with high basal FSH concentrations have a poor outcome based on the low quality of the oocytes retrieved. However, young low responders with normal day 3 FSH concentrations, although their ovarian reserve may be compromised (Pellicer et al., 1998), might benefit from alternative protocols such as the stop protocol, as their chances of achieving a pregnancy seem to be similar to those of normal responders (Lashen et al., 1999
).
It is concluded that low responders with normal basal FSH concentrations may benefit from increasing the doses of gonadotrophins in order to proceed to oocyte retrieval and avoid a new cancelled cycle, with the financial and emotional implications and frustration for both the patient and physician. The stop protocol increases the number of available embryos and decreases the quantity of gonadotrophins required. Further studies are required to determine whether early cessation of GnRHa leads to an increase in LH concentrations in the late induction phase, and subsequently to decreased implantation.
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Notes |
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References |
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Beckers, N.G.M., Laven, J.S.E., Eijkemans, M.J.C. et al. (2000) Follicular and luteal phase characteristics following early cessation of gonadotrophin-releasing hormone agonist during ovarian stimulation for in-vitro fertilization. Hum. Reprod., 15, 4349.
Bhal, P.S., Pugh, N.D., Chui, D.K. et al. (1999) The use of transvaginal power Doppler ultrasonography to evaluate the relationship between perifollicular vascularity and outcome in in-vitro fertilization treatment cycles. Hum. Reprod., 14, 939945.
Chong, A., Rafael, R. and Forte, C. (1986) Influence of weight in the induction of ovulation with human menopausal gonadotropin and human chorionic gonadotropin. Fertil. Steril., 49, 599603.
Crosignani, P., Ragni, G., Lombroso, G. et al. (1989) IVF: induction of ovulation in poor responders. J. Steroid Biochem., 32, 171173.[ISI][Medline]
Dirnfield, M., Fruchter, O., Yshai, D. et al. (1999) Cessation of gonadotrophin-releasing hormone analogue (GnRH-a) upon down-regulation versus conventional long GnRH-a protocol in poor responders undergoing in vitro fertilization. Fertil. Steril., 72, 406411.[ISI][Medline]
Engmann, L., Sladkevicius, P., Agrawal, R. et al. (1999) Value of ovarian stromal blood flow velocity measurement after pituitary suppression in the prediction of ovarian responsiveness and outcome of in vitro fertilization treatment. Fertil. Steril., 71, 2229.[ISI][Medline]
Faber, B., Mayer, J., Cox, B. et al. (1998) Cessation of gonadotrophin-releasing hormone agonist therapy combined with high-dose gonadotrophin stimulation yields favorable pregnancy results in low responders. Fertil. Steril., 69, 826830.[ISI][Medline]
Garcia, J.E., Jones, G.S., Acosta, A.A. et al. (1983) Human menopausal gonadotrophin/human chorionic gonadotrophin follicular maturation for oocyte aspiration: phase II, 1981. Fertil. Steril., 39, 174179.[ISI][Medline]
Gelety., T., Pearlstone, A. and Surrey, E. (1995) Short-term endocrine response to gonadotropin-releasing hormone agonist initiated in the early follicular, mid luteal or late luteal phase in normally cycling women. Fertil. Steril., 64, 10741080.[ISI][Medline]
Gil-Salom, M., Mínguez, Y., Rubio, C. et al. (1995) Efficacy of intracytoplasmic sperm injection using testicular spermatozoa. Hum. Reprod., 10, 31663170.[Abstract]
Horvath, P.M., Styler, M., Hammond, J.M. et al. (1988) Exogenous gonadotropin requirements are increased in leuprolide suppressed women undergoing ovarian stimulation. Fertil. Steril., 49, 159162.[ISI][Medline]
Karande, V. and Gleicher, N. (1999) A rational approach to the management of low responders in in-vitro fertilization. Hum. Reprod., 14, 17441748.
Kowalik, A., Barmat, L., Damario, M. et al. (1998) Ovarian estradiol production in vivo. Inhibitory effect of leuprolide acetate. J. Reprod. Med., 43, 413417.[ISI][Medline]
Land, J., Yarmolinskaya, M., Dumoulin, J. et al. (1996) High-dose human menopausal gonadotropin stimulation in poor responders does not improve in vitro fertilization outcome. Fertil. Steril., 65, 961965.[ISI][Medline]
Lashen, H., Ledger, W., Lopez-Bernal, A. et al. (1999) Poor responders to ovulation induction: is proceeding to in-vitro fertilization worthwhile? Hum. Reprod., 14, 964969.
Latouche, J., Crumeyrolle-Arias, M., Jordan, D. et al. (1989) GnRH receptors in human granulosa cells: anatomical localization and characterization by autoradiographic study. Endocrinology, 125, 17391741.[Abstract]
Laufer, N., De Cherney, A.H., Tarlatzis, B.C. et al. (1986) The association between pre-ovulatory serum 17-ß estradiol pattern and conception in human premenopausal gonadotrophin-human chorionic gonadotrophin stimulation. Fertil. Steril., 46, 7376.[ISI][Medline]
Pellicer, A., Lightman, A., Diamond, M.P. et al. (1987) Outcome of in vitro fertilization in women with low response to ovarian stimulation. Fertil. Steril., 47, 812815.[ISI][Medline]
Pellicer, A., Simón, C., Miró, F. et al. (1989) Ovarian response and outcome of in-vitro and fertilization in patients treated with gonadotrophin-releasing hormone analogues in different phases of the menstrual cycle. Hum. Reprod., 4, 285289.[Abstract]
Pellicer, A., Ardiles, G., Neuspiller, F. et al (1998) Evaluation of the ovarian reserve in young low responders with normal basal levels of follicle-stimulating hormone using three-dimensional ultrasonography. Fertil. Steril., 70, 671675.[ISI][Medline]
San Roman, G., Surrey, E., Judd, H. et al. (1992) A prospective randomized comparison of luteal phase versus concurrent follicular phase initiation of gonadotropin-releasing hormone agonist for in vitro fertilization. Fertil. Steril., 58, 744749.[ISI][Medline]
Scott, R.T. (1996) Evaluation and treatment of low responders. Semin. Reprod. Endocrinol., 14, 317337.[ISI][Medline]
Scott, R.T., Hofmann, G.E., Oehninger, S. et al. (1990) Intercycle variability of day 3 follicle-stimulating hormone levels and its effect on stimulation quality in in vitro fertilization. Fertil. Steril., 54, 297302.[ISI][Medline]
Simón, C., Garcia-Velasco, J.A., Valbuena, D. et al. (1998) Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil. Steril., 70, 234239.[ISI][Medline]
Sungurtekin, U. and Jansen, R.P.S. (1995) Profound luteinizing hormone suppresion after stopping the gonadotrophin releasing hormone-agonist leuprolide acetate. Fertil. Steril., 63, 663665.[ISI][Medline]
Valbuena, D., Pellicer, A., Guanes, P.P. et al. (1997) Effect of disruption versus continuation of gonadotrophin-releasing agonist after human chorionic gonadotrophin administration on corpus luteum function in patients undergoing ovulation induction for in-vitro fertilization. Hum. Reprod., 12, 21182122.[Abstract]
Van-Hoof, M.H., Alberda, A.T., Huisman, G.J. et al. (1993) Doubling the human menopausal gonadotrophin dose in the course of an in-vitro fertilization treatment cycle in low responders: a randomized study. Hum. Reprod., 8, 369373.[Abstract]
Zeleznik, A.J., Schuler, H.M. and Reichert, L.E.J. (1981) Gonadotrophin-binding sites in the Rhesus monkey ovary: role of the vasculature in the selective distribution of human chorionic gonadotrophin to the preovulatory follicle. Endocrinology, 109, 356362.[Abstract]
Submitted on April 10, 2000; accepted on August 2, 2000.