1 Centre for Reproductive Medicine, Dutch-Speaking Free University of Brussels, Brussels, Belgium
Abstract
Luteinizing hormone (LH) is mandatory for the maintenance of the corpus luteum. Ovarian stimulation for IVF has been associated with a defective luteal phase. The luteal phases of two groups of patients with normal menstrual cycles and no endocrinological cause of infertility were retrospectively analysed in IVF cycles. Thirty-one infertile patients stimulated with human menopausal gonadotrophins (HMG) for IVF to whom the gonadotrophin-releasing hormone (GnRH) antagonist Cetrorelix 0.25 mg was also administered to prevent the LH surge (group I) were compared with 31 infertile patients stimulated with HMG alone (group II). Despite differences in the stimulation outcome, luteal LH serum concentrations were similar in the two groups. LH values dropped from 2.3 ± 1 IU/l on the day of human chorionic gonadotrophin (HCG) administration to 1.1 ± 0.7 IU/l on day HCG +2 in group I (P < 0.0001) and from 5.1 ± 3 to 1.2 ± 1.7 IU/l (P < 0.0001) in group II. In the mid-luteal phase, LH concentrations were low in both groups. Our results suggest that suppressed LH concentrations in the early and mid-luteal phase may not be attributed solely to the GnRH-antagonist administration. Pituitary LH secretion may be inhibited by supraphysiological steroid serum concentrations via long-loop feedback and/or by the central action of the exogenously administered HCG via a short-loop mechanism.
Key words: GnRH antagonist/IVF/LH/luteal phase/ovarian stimulation
Introduction
The administration of gonadotrophin-releasing hormone antagonist (GnRH) Cetrorelix has been shown to be effective in blocking the luteinizing hormone (LH) surge in ovarian stimulation cycles for IVF (Diedrich et al., 1994; Albano et al., 1996
; Felberbaum et al., 1996
; Albano et al., 1997
).
In contradiction to GnRH-agonist cycles, where pituitary function remains impaired for the entire length of the luteal phase after the arrest of the agonist (Smitz et al., 1992), adenohypophysis maintains its responsiveness to endogenous GnRH stimulus after antagonist treatment (Felberbaum et al., 1995
) and it was suggested that antagonist cycles may not be in need of luteal phase support (Albano et al., 1997
). Nevertheless, an impaired luteal phase in terms of duration and/or serum progesterone concentrations was observed in four out of six patients stimulated with the association of human menopausal gonadotrophin (HMG) and Cetrorelix 0.5 mg cycles with no luteal phase supplementation (Albano et al., 1998
). A further analysis of the luteal phases in Cetrorelix 0.25 or 0.5 mg/HMG cycles showed that LH serum concentrations were reduced to almost undetectable levels two days after the human chorionic gonadotrophin (HCG) injection and for the whole length of the luteal phase (Albano et al., 1999
).
As LH is mandatory for the maintenance and normal steroidogenic activity of the human corpus luteum (Casper and Yen, 1979; Schriock et al., 1985
; Mais et al., 1986
), abnormal LH secretion may account for a defective luteal phase. The aim of this study is to investigate further the possible causes of the observed decrease in serum LH concentrations after GnRH-antagonist treatment, by comparing patients stimulated with the association of HMG and the antagonist Cetrorelix 0.25 mg with patients stimulated with HMG only, for IVF.
Materials and methods
Two groups of patients were compared in this retrospective analysis. In both groups, the detection of infertility caused by endocrinopathies or polycystic ovarian syndrome or the appearance of polycystic ovaries in the ultrasound scan were main exclusion criteria. Patients with premature LH rise (e.g. two consecutive measurements of LH >10 IU/l) (Devroey et al., 1995) were also excluded from the analysis.
In the first group, 31 subjects were stimulated with a combination of HMG and the antagonist Cetrorelix 0.25mg. The stimulation protocol has been previously described in detail (Albano et al., 1996, 1997
). In brief, subjects were infertile women between 23 and 37 years of age, with regular menstrual cycles (2435 days) undergoing IVF in five cycles (16%) or intracytoplasmic sperm injection (ICSI) in 26 cycles (84%). Controlled ovarian stimulation was carried out with three ampoules (225 IU) HMG (Humegon; Organon, Oss, The Netherlands and Menogon; Ferring, Kiel, Germany) starting on day 2 of the menstrual cycle. The dose was adjusted individually from day 6 of the treatment according to oestradiol values and ultrasonographic follicular measurements. From day 6 of the HMG injection onwards (day 7 of the menstrual cycle), 0.25 mg of Cetrorelix (ASTA Medica AG, Frankfurt Main, Germany) were also administered s.c. in the anterior abdominal wall, up to and including the last day of HMG administration. Ovulation was induced when at least three follicles were
17 mm in diameter, by the injection of 10.000 IU of HCG (Pregnyl; Organon Oss, The Netherlands). A maximum of three embryos was replaced 2 days after the oocyte retrieval. All the subjects received luteal-phase support by means of one injection of 1500 IU of HCG every three days starting on the day of embryo transfer.
The second group consisted of 31 infertile patients stimulated with HMG for IVF. The stimulation protocol has also been previously described in detail (Devroey et al., 1995). In brief, patients were 2536 years of age with regular menstrual cycles. Controlled ovarian stimulation was carried out with three ampoules (225 IU) HMG (Normegon; Organon Oss, The Netherlands and Metrodin; Serono, Geneva, Switzerland) starting on day 2 or 3 of the menstrual cycle. After 4 days of treatment the dose was adjusted individually according to serum oestradiol concentrations and ultrasound follicular measurements. Ovulation was induced by 10 000 IU of HCG (Pregnyl; Organon Oss, the Netherlands) when three follicles
17 mm were detected by ultrasonography. The luteal phase was supplemented with HCG 1500 IU every 3 days starting 2 days after the embryo transfer.
In both groups, intensive hormonal monitoring was performed through daily blood samples during the peri-ovulatory period for the detection of a premature LH surge until the day of embryo transfer. Serum gonadotrophins were measured by specific monoclonal immunoradiometric assays (IRMA) for follicle stimulating hormone (FSH) and LH and expressed in IU/l (conversion factor to SI unit 1.00; First International Reference preparation for LH 68/40 and Second International Reference preparation for FSH 78/549). The LH assay had a sensitivity of 0.3 IU/l and within- and between-assay coefficients of variation of 7 and 9% respectively. Steroid serum concentrations were expressed in ng/l for oestradiol and µg/l for progesterone (conversion factor to SI unit 3.671 for oestradiol and 3.180 for progesterone).
Clinical pregnancy was defined as the presence of a gestational sac in the ultrasound scan at 7 weeks. Data were analysed by means of a Wilcoxon rank sum test using MedCalc software statistical program (MedCalc Software; Mariakerke, Belgium). Values are expressed as mean ± SD. Statistical significance was defined as a P value of <0.05.
Results
Patients' age and day 3 FSH levels were comparable in both groups. In group I (HMG/Cetrorelix 0.25 group) the mean age of the patients was 30.7 ± 4.1 years and in group II (HMG group) it was 30.3 ± 2.5 years. Although cycle characteristics were different between the two groups, the number of transferred embryos was similar (2.7 ± 0.4 in the HMG/Cetrorelix 0.25 mg and 2.5 ± 0.5 in the HMG group), resulting in eight clinical pregnancies per group (pregnancy rate/embryo transfer of 25.8%) (Table I).
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Cycle characteristics were different between the two stimulation protocols. In the HMG/Cetrorelix 0.25 mg group, ovarian stimulation was longer, the number of administered ampoules and the number of retrieved oocytes were significantly higher. This may be explained by the fact that as GnRH antagonists successfully prevent a premature LH surge, ovarian stimulation may be prolonged more than in cycles treated without antagonists, in order to obtain a larger number of mature oocytes.
In the pre-ovulatory phase, LH serum concentrations were lower in the antagonist group, as antagonist treatment significantly reduces LH levels (Leroy et al., 1994). One day after the ovulatory HCG, LH serum concentrations dropped in the HMG group but not in the Cetrorelix group, probably due to the arrest of the antagonist. Despite the differences in cycle characteristics between the two protocols, the luteal phase LH serum concentrations were similar from day 2 following the ovulatory HCG onwards. It has been previously reported from our group that treatment with the association of HMG and Cetrorelix 0.25 or 0.5 mg for ovarian stimulation reduces LH serum concentrations to almost undetectable levels for the whole length of the luteal phase in cycles supplemented with HCG (Albano et al., 1999
). Similarly, in GnRH-agonist/HMG cycles, undetectable LH levels and low progesterone secretion in the luteal phase have been described, accounting for a luteal-phase defect and making luteal-phase support necessary (Smitz et al., 1988
). In GnRH-agonist cycles, these low LH levels may be attributed to a prolonged impairment of the pituitary gonadotrophin secretory capacity after GnRH- agonist treatment (Smitz et al., 1992
). On the contrary, the pituitary remains responsive to GnRH after antagonist treatment (Gordon et al., 1990
; Felberbaum et al., 1995
) and normal corpus luteum function is preserved after mid-follicular antagonist administration (Mais et al., 1986
). Since, in our results, LH values were strikingly similar in the early and mid-luteal phase in the two groups, it may be postulated that this observed decrease in LH serum concentrations may not be attributed solely to the GnRH antagonist administration.
Progesterone modulates LH secretion during the luteal phase by influencing the LH pulse amplitude and pituitary release of LH (Soules et al., 1984). A short exposure to physiological levels of progesterone, in the range of the early luteal phase, has a stimulatory effect on LH secretion by acting directly at the pituitary level (Couzinet et al., 1992
; Couzinet and Schaison, 1993
). On the other hand, a longer exposure to progesterone or the combined action of oestrogen and progesterone, results in reduced frequency of LH secretion by a possible action at the hypothalamic level (Steele and Judd, 1986
, 1988
; Nippoldt et al., 1989
). Consequently, as ovarian stimulation results in supraphysiological steroid serum concentrations as compared to natural cycles, it may be postulated that these high steroid serum concentrations may adversely effect the LH secretion by disturbing the feedback mechanisms.
The fact that ovarian stimulation reduces LH concentrations in the luteal phase and results in a luteal-phase defect has also been previously proposed (Messinis and Templeton, 1987). In cycles stimulated with FSH, lower early serum LH concentrations have been detected than in natural cycles, or in natural cycles supplemented with exogenous oestrogen (Messinis and Templeton, 1987
). Similarly, in cycles stimulated with clomiphene citrate/HMG/HCG for IVF, serum LH concentrations decreased from 20 IU/l (the day after the HCG injection) to a nadir of 35 IU/l in the mid-luteal phase (Smitz et al., 1988
).
As well as supraphysiological steroid concentrations in ovarian stimulation, there is one more possible mechanism that might further affect pituitary LH secretion. A long time ago it was suggested that a negative short-loop feedback mechanism might exist which controls LH secretion (David et al., 1966). According to this hypothesis, HCG may affect pituitary LH secretion negatively, by reducing hypothalamic GnRH due to its similarity to the LH molecule but also due to its longer half-life (Damewood et al., 1989
). Although in animal studies such a negative feedback exists (Silverman et al., 1981
; Patritti- Laborde et al., 1982
) there is a debate in the literature about its existence in humans, with some of the studies supporting this hypothesis (Miyake et al., 1978
, 1979
) and others not (Kyle et al., 1989
; Nader and Berkowitz, 1992
). Nevertheless, recent findings in in-vitro studies further support this idea. GT1-7 neurons which are morphologically and functionally similar to GnRH neurons were found to contain LH/HCG receptors (Lei and Rao, 1994
). In addition, exogenously administered HCG was found to decrease the expression of GnRH receptor gene in GT1-7 cells (Li et al., 1996
) or GnRH secretion in immortalized GnRH neurons (Mores et al., 1996
).
In stimulated cycles this assumption was supported by Demoulin et al. (1991). As mid-luteal LH serum concentrations were significantly lower in stimulated cycles compared to control natural cycles, the authors postulated that this might be due a possible short negative feedback from the exogenously administered ovulatory HCG. Furthermore, in cycles stimulated with HMG/Cetrorelix 0.5 mg and receiving no luteal phase supplementation, LH concentrations started to increase 8 days after the HCG (Albano et al., 1998), which coincides with the time that exogenous HCG is cleared from the circulation (Mannaerts et al., 1998
). On the contrary, LH concentrations remained low in cycles supplemented with HCG (Albano et al., 1999
). As, in GnRH antagonist cycles, triggering of ovulation is possible with a GnRH-agonist (Olivennes et al., 1996
), the effect that alternative methods to induce the final oocyte maturation (e.g. GnRH-agonist or recombinant LH) exert on the luteal phase, remains be investigated.
In conclusion, ovarian stimulation with virtually all the currently used stimulation protocols results in reduced LH serum concentrations in the early and mid-luteal phase. These low LH serum concentrations may contribute to the luteal phase defect observed after ovarian stimulation. Supraphysiological steroid serum concentrations may interfere with LH secretion via long-loop feedback, but, additionally, the exogenously administered HCG might amplify LH secretion arrest via a second short-loop negative feedback.
Notes
2 To whom correspondence should be addressed at: AZ-VUB, Centre for Reproductive Medicine, Laarbeeklaan 101, 1090 Brussels, Belgium. E-mail: paul.devroey{at}az.vub.ac.be or mtavaniotou{at}hotmail.com
References
Albano, C., Smitz, J., Camus, M. et al. (1996) Hormonal profile during the follicular phase in cycles stimulated with a combination of human gonadotrophin and gonadotrophin-releasing hormone antagonist (Cetrorelix). Hum. Reprod., 11, 21142118.[Abstract]
Albano, C., Smitz, J., Camus, M., et al. (1997) Comparison of different doses of gonadotropin-releasing hormone antagonist Cetrorelix during controlled ovarian hyperstimulation. Fertil. Steril., 67, 917922.[ISI][Medline]
Albano, C., Grimbizis, G., Smitz, J., et al. (1998) The luteal phase of nonsupplemented cycles after ovarian superovulation with human menopausal gonadotropin and the gonadotropin-releasing hormone antagonist Cetrorelix. Fertil. Steril., 70, 357359.[ISI][Medline]
Albano, C., Smitz, J., Tournaye, H., et al. (1999) Luteal phase and clinical outcome after human menopausal gonadotrophin/gonadotrophin releasing hormone antagonist treatment for ovarian stimulation in in-vitro fertilization/intracytoplasmic sperm injection cycles. Hum. Reprod., 14, 14261430.
Casper, R.F. and Yen, S.S.C. (1979) Induction of luteolysis in the human with a long-acting analog of luteinizing hormone-releasing factor. Science, 205, 408410.[ISI][Medline]
Couzinet, B., Brailly, S., Bouchard, P. et al. (1992) Progesterone stimulates luteinizing hormone secretion by acting directly on the pituitary. J. Clin. Endocrinol. Metab., 74, 374378.[Abstract]
Couzinet, B. and Schaison, G. (1993) The control of gonadotrophin secretion by ovarian steroids. Hum. Reprod., 8 (Suppl. 2), 97101.[Abstract]
Damewood, M.D., Shen, W., Zacur, H.A. et al. (1989) Disappearance of exogenously administered human chorionic gonadotropin. Fertil. Steril., 52, 398400.[ISI][Medline]
David, M.A., Fraschini, F. and Martini, L. (1966) Control of LH secretion: role of a `short' feedback mechanism. Endocrinology, 78, 5569.[ISI][Medline]
Demoulin, A., Dubois, M., Gerday, C. et al. (1991) Variations of luteinizing hormone serum concentrations after exogenous human chorionic gonadotropin administration during ovarian hyperstimulation. Fertil. Steril., 55, 797780.[ISI][Medline]
Devroey, P., Tjandraprawira, K., Mannaerts, B. et al. (1995) A randomized, assessor-blind, group comparative efficacy study to compare the effects of Normegon® and Metrodin® in infertile female patients undergoing in-vitro fertilization. Hum. Reprod., 10, 332337.[Abstract]
Diedrich, K., Diedrich, C., Santos, E. et al. (1994) Suppression of the endogenous luteinizing hormone surge by the gonadotrophin-releasing hormone antagonist Cetrorelix during ovarian stimulation. Hum. Reprod., 9, 788791.[Abstract]
Felberbaum, R., Reissmann, T., Küpker, W. et al. (1995) Preserved pituitary response under ovarian stimulation with HMG and GnRH antagonists (Cetrorelix) in women with tubal infertility. Eur. J. Obstet. Gynecol. Reprod. Biol., 61, 151155.[ISI][Medline]
Felberbaum, R.E., Reissmann, T., Küpker, W. et al. (1996) Hormone profiles under ovarian stimulation with human menopausal gonadotropin (HMG) and concomitant administration of the gonadotropin releasing hormone (GnRH)-antagonist Cetrorelix at different dosages. J. Assist. Reprod. Genet., 13, 216222.[ISI][Medline]
Gordon, K., Williams, R.F., Danforth, D.R. et al. (1990) A novel regimen of gonadotropin-releasing hormone (GnRH) antagonist plus pulsatile GnRH: controlled restoration of gonadotropin secretion and ovulation induction. Fertil. Steril., 54, 11401145.[ISI][Medline]
Kyle, C.V., Griffin, J., Jarrett, A. et al. (1989) Inability to demonstrate an ultrashort loop feedback mechanism for luteinizing hormone in humans. J. Clin. Endocrinol. Metab., 69, 170176.[Abstract]
Lei, Z.M. and Rao, Ch.V. (1994) Novel presence of luteinizing hormone/human chorionic gonadotropin (HCG) receptors gene and the down-regulating action of HCG on gonadotropin-releasing hormone gene expression in immortalized hypothalamic GT1-7 neurons. Mol. Endocrinol., 8, 11111121.[Abstract]
Leroy, I., d'Acremont, M.F., Brailly-Tabard, S. et al. (1994) A single injection of a gonadotropin-releasing hormone (GnRH) antagonist (Cetrorelix) postpones the luteinizing hormone (LH) surge: further evidence for the role of GnRH during the LH surge. Fertil. Steril., 62, 461467.[ISI][Medline]
Li, X., Lei, Z.M. and Rao, Ch.V. (1996) Human chorionic gonadotropin down-regulates the expression of gonadotropin-releasing hormone receptor gene in GT1-7 neurons. J. Clin. Endocrinol. Metab., 137, 899904.
Mannaerts, B.M., Geurts, T.B. and Odnik, J. (1998) A randomized three-way cross over study in healthy pituitary-suppressed women to compare the bioavailability of human chorionic gonadotrophin (Pregnyl) after intramuscular and subcutaneous administration. Hum. Reprod., 13, 14611464.[Abstract]
Mais, V., Kazer, R.R., Cetel, N.S. et al. (1986) The dependency of folliculogenesis and corpus luteum function on pulsatile gonadotropin secretion in cycling women using a gonadotropin-releasing hormone antagonist as a probe. Fertil. Steril., 62, 12501255.
Messinis, I.E. and Templeton, A.A. (1987) Disparate effects of endogenous and exogenous oestradiol on luteal phase function in women. J. Reprod. Fertil., 79, 549554.[Abstract]
Mores, N., Krsmanovic, L.Z. and Catt, K.J. (1996) Activation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptidase release. Endocrinology, 137, 57315734.[Abstract]
Miyake, A., Aono, T., Kinugasa, T. et al. (1978) The time course change after castration in short-loop negative feedback control of LH by HCG in women. Acta Endocrinol. (Copenh.), 88, 16.[Medline]
Miyake, A., Aono, T., Kinugasa, T. et al. (1979) Suppression of serum levels of luteinizing hormone by shortand long-loop negative feedback in ovariectomized women. J. Endocrinol., 80, 353356.[Abstract]
Nader, S. and Berkowitz, A.S. (1992) Endogenous luteinizing hormone surges following administration of human chorionic gonadotropin: further evidence for lack of loop feedback in humans. J. Assist. Reprod. Genet., 9, 124127.[ISI][Medline]
Nippoldt, T.B., Reame, N., Kelch, R.P. et al. (1989) The roles of estradiol and progesterone in decreasing luteinizing hormone pulse frequency in the luteal phase of the menstrual cycle. J. Clin. Endocrinol. Metab., 69, 6776.[Abstract]
Olivennes, F., Fanchin, R., Bouchard, F. et al. (1996) Triggering of ovulation by a gonadotropin-releasing hormone (GnRH) agonist in patients pre-treated with a GnRH antagonist. Fertil. Steril., 66, 151153.[ISI][Medline]
Patritti-Laborde, N., Asch, R.H., Pauerstein, C.J. et al. (1982) Prevention of the postcoital luteinizing hormone surge by ultrashort feedback control. Fertil. Steril., 38, 349353.[ISI][Medline]
Schriock, E., Monroe, S., Martin, M. et al. (1985) Effect on corpus luteum function of luteal phase administration of a potent gonadotropin-releasing analogue (nafarelin). Fertil. Steril., 43, 844850.[ISI][Medline]
Silverman, A.Y., Smith, C.G., Siler-Khodr, T.M. et al. (1981) Human chorionic gonadotropin blocks the estrogen-induced luteinizing hormone release in long-term castrated rhesus monkeys: evidence for an ultrashort-loop negative feedback. Fertil. Steril., 35, 7478.[ISI][Medline]
Smitz, J., Devroey, P., Camus, M. et al. (1988) The luteal phase and early pregnancy after combined GnRH-agonist/HMG treatment for superovulation in IVF or GIFT. Hum. Reprod., 3, 585590.[Abstract]
Smitz, J., Erard, P., Camus, M. et al. (1992) Pituitary gonadotrophin secretory capacity during the luteal phase in superovulation using GnRH-agonists and HMG in a desensitization or flare-up protocol. Hum. Reprod., 7, 12251229.[Abstract]
Steele, P.A. and Judd, S.J. (1986) Role of endogenous opioids in reducing the frequency of pulsatile luteinizing hormone secretion induced by progesterone in normal women. Clin. Endocrinol. (Oxf.), 25, 669674.[ISI][Medline]
Steele, P.A and Judd, S.J. (1988) Positive and negative feed-back effect of progesterone on luteinizing hormone secretion in post-menopausal women. Clin. Endocrinol., 29, 17.[ISI][Medline]
Soules, M., Steiner, R., Clifton, D. et al. (1984) Progesterone modulation of pulsatile luteinizing hormone secretion in normal women. J. Clin. Enocrinol. Metab., 58, 378382.[Abstract]
Submitted on August 29, 2000; accepted on January 4, 2001.