Reproductive Endocrinology Centre, University of Bologna, Bologna, Italy
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Abstract |
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Key words: follicle-stimulating hormone/human chorionic gonadotrophin/luteinizing hormone/ovarian follicle/ovulation induction
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Introduction |
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In our previous work we extensively analysed the effect of standard HMG regimens as well as the supplementation of FSH with low-dose HCG in order to test the effects of LH:FSH activity ratios greater than 1:1 (Filicori et al., 1999a,b
, 2002
). Conversely, many clinical centres performing assisted reproduction techniques commonly resort to the combination of different gonadotrophin preparations yielding lower LH:FSH ratios than found in traditional HMG (1:1); nevertheless, the precise impact of these regimens on folliculogenesis and steroidogenesis has never been prospectively assessed. Thus, in the current investigation we elected to investigate thoroughly the other side of the regimen spectrum of HMG treatment, i.e. the impact of progressively declining ratios of LH:FSH activity. In this study we elected to treat patients with a long GnRH agonist regimen maximally to reduce interference from endogenous gonadotrophins; furthermore, exogenous gonadotrophins were administered at a fixed dose for at least 14 consecutive days to limit the confounding effects of variable drug dosages. Thus, the drug regimens we employed in this study are not standard for patient candidates for intrauterine insemination (IUI) and were not meant to assess the effect of different FSH:LH ratios on pregnancy rates and type of gestation in this clinical setting. Nevertheless, results of this study provided invaluable information on LH and FSHregulated follicular development and ovarian steroid secretion and can be used better to understand and optimize gonadotrophin ovulation induction regimens.
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Materials and methods |
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Protocol
Our Institutional Review Board approved the protocol and all patients provided informed consent. Patients underwent early follicular phase reproductive hormone determinations and were then randomly assigned to four age- and weight-matched groups. The incidence of patients who had previously undergone gonadotrophin ovulation induction as well as cause of infertility were similar in all groups. Patients were not blinded to treatment, which was started in the mid-luteal phase of a spontaneous menstrual cycle with the administration of a single injection of 3.75 mg of depot triptorelin (Decapeptyl® 3.75; Ipsen S.p.A., Milan, Italy). Ovulation induction began 14 days thereafter. Menotrophin preparations used included highly purified (HP) FSH containing FSH 75 IU/ampoule and negligible LH activity levels (Metrodin® HP; Serono Pharma S.p.A., Rome, Italy) and HMG containing FSH 75 IU and LH activity 75 IU/ampoule (Menogon®; Ferring S.p.A., Milan, Italy). In order to obtain non-standard LH:FSH activity ratios these menotrophin preparations were mixed by a physician before i.m. administration. Each treatment group consisted of 30 patients. The gonadotrophin regimens adopted in each group were as follows: group A, FSH 150 IU daily only; group B, FSH 150 IU and LH activity 37.5 IU daily; group C, FSH 150 IU and LH activity 75 IU daily; group D, FSH 150 IU and LH activity 150 IU daily. In all patients gonadotrophins were administered at 16001800 h and the menotrophin dose was not changed for 14 days or until at least four ovarian follicles of >14 mm diameter and estradiol (E2) levels of 8001500 pg/ml were detected (final maturation parameters). If these parameters were not achieved by the 14th day of treatment, increments uniquely of FSH activity (not of LH activity) were allowed with the following schedule: FSH 225 IU daily on days 1517; FSH 300 IU daily thereafter. When the final maturation parameters were attained, 10000 IU of HCG were administered to trigger ovulation and IUI with a sperm swim-up procedure was performed 36 h thereafter. The luteal phase was supported with 90 mg daily of intravaginal progesterone gel (Crinone®; Serono) administered from days 314 day following the pre-ovulatory HCG dose.
Monitoring
Treatment monitoring was conducted throughout menotrophin administration. Each day one blood sample was drawn at 08000900 h in a standard manner, and two serum aliquots were obtained: E2 was measured daily in one of the serum aliquots for clinical monitoring while the second aliquot was stored at 20°C for later measurements of LH, FSH, E2, progesterone, testosterone, and HCG. Transvaginal pelvic ultrasound was performed on menotrophin treatment days 0 and 6 and at alternate days thereafter, until pre-ovulatory HCG administration. The physician performing pelvic ultrasound was blinded as to which arm of the protocol each patient belonged.
Hormone assays
Luteinizing hormone, FSH, E2, progesterone, testosterone, and HCG were measured with chemiluminescence assays (Chiron Diagnostics ACS 180, Milan, Italy). The minimal detectable level (MDL) of LH was 0.1 IU/l; the interassay coefficient of variation (CV) at low, intermediate, and high levels of the standard curve were 4.1, 7.8, and 4.5% respectively. The in-vitro addition of up to 200 000 IU/l of HCG did not affect LH determinations in this assay, as assessed at multiple levels of the standard curve. The MDL of FSH was 0.3 IU/l; the interassay CV at low, intermediate, and high levels of the standard curve were 3.9, 4.0, and 3.9% respectively. The MDL of E2 was 10 pg/ml: the interassay CV at low, intermediate, and high levels of the standard curve were 5.4, 3.7, and 13.1% respectively. The MDL of progesterone was 0.1 ng/ml; the interassay CV at low, intermediate, and high levels of the standard curve were 7.0, 3.9, and 6.7% respectively. The MDL of testosterone was 0.1 ng/ml; the interassay CV at low, intermediate, and high levels of the standard curve were 5.1, 5.1, and 4.9% respectively. The MDL of HCG in this ß-specific assay was 0.1 IU/l.; the interassay CV at low, intermediate, and high levels of the standard curve were 6.9, 3.1, and 2.2% respectively. The in-vitro addition of up to 200 IU/l of LH did not affect HCG determinations in this assay, as assessed at multiple levels of the standard curve.
Statistical evaluation
Data were expressed as mean ± SE. Serum hormone levels were calculated in each cycle as area under the curve (AUC). Between-group differences of continuous variables were assessed with one way analysis of variance (ANOVA) with Tukey test or KruskalWallis ANOVA on ranks with the Dunn's method, as appropriate. Correlations were assessed with the Pearson product moment correlation. Between-group differences in non-continuous variables were assessed with the 2 method with the Yates' correction, if needed.
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Results |
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Discussion |
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Our group and others had previously observed that the LH activity provided by HMG preparations consists of both LH and HCG (Stokman et al., 1993; Rodgers et al., 1994
; Filicori et al., 2001
). HCG has a longer half-life and is thus more potent than LH (about six times) (Stokman et al., 1993
); nevertheless, all physiological and pharmacological actions of HCG are exerted through LH receptors as separate HCG receptors have never been identified. Pharmaceutical companies do not indicate the specific amounts of LH and HCG contained in each ampoule/batch of HMG; nevertheless, the gonadotrophin content of HMG is precisely titrated to provide 75 units of LH activity (consisting of both LH and HCG) in each ampoule. In this study HCG was detected in the serum of several patients (Figure 1
) and we found that the dose of LH activity administered better correlated to serum HCG (r = 0.448, P < 0.00005) than to serum LH levels (r = 0.235, P < 0.01). Thus, far from being an unwanted contaminant of menotrophin preparations, HCG may play an important role in gonadotrophin ovulation induction and in the modulation of ovarian follicle function. Because of its prolonged half-life, HCG may be better suited than LH to provide longer-lasting and more stable stimulation of granulosa cell LH receptors. We also confirmed that the addition of LH activity permits shortening of the duration of COS (Filicori et al., 1999a
,b
, 2001
). This effect was significant when at least 75 IU daily of LH activity were employed in the current study (Table II
) and it is likely to be due to the stimulatory action exerted by LH activity on the granulosa cells of larger ovarian follicles once LH receptors are expressed by these cells in the mid-late follicular phase (Zeleznik et al., 1974
; Shima et al., 1987
). Conversely, the occurrence of small (<10 mm) ovarian follicles was significantly reduced in all the patients receiving some degree of LH activity stimulation; the number of small pre-ovulatory follicles progressively declined as the amount of LH activity used in each group increased (Figure 2
). A dose-related effect of LH activity on the demise of small follicles was confirmed by the finding of a highly significant inverse correlation between pre-ovulatory small follicles and both LH activity dose administered and follicular phase serum HCG concentrations (Figure 4
). This effect of LH activity may be mediated by increased ovarian androgen production (Louvet et al., 1975
; Filicori et al., 1996
) as the LH/HCG dose administered was positively correlated to serum testosterone levels (Figure 3B
). No correlation was found between follicular phase serum LH levels and follicle development; this finding further emphasizes the concept that the HCG component of HMG LH activity may critically contribute to overall menotrophin efficacy. The negative relationship we found between LH activity dose administered and small pre-ovulatory follicle development confirms and extends our observations (Filicori et al., 2001
, 2002
) and preliminary reports by other groups (Arguinzoniz et al., 2000
; Loumaye et al., 2000
) indicating that LH activity can selectively modulate follicle maturation at different stages of development. One of the key parameters that predispose patients undergoing ovulation induction and COS to develop OHSS is the occurrence of small pre-ovulatory ovarian follicles (Blankstein et al., 1987
). Thus, as we recently proposed (Filicori and Cognigni, 2001
) the use of variable LH:FSH ratios at different stages of COS could be employed to limit markedly the occurrence of this potentially dangerous group of ovarian follicles.
Moderate progesterone increments in the follicular phase, so-called premature follicle luteinization, have been frequently reported during COS (Silverberg et al., 1991; Fanchin et al., 1993
) and were thought to derive from excessive granulosa cell stimulation caused by untimely increments of endogenous or exogenous LH activity; it was also suggested that the HCG content of HMG could be pivotal in triggering this phenomenon (Copperman et al., 1995
). Nevertheless, profound suppression of endogenous LH secretion with GnRH agonists (Hofmann et al., 1993b
; Ubaldi et al., 1996b
) or GnRH antagonists (Ubaldi et al., 1996a
) and the use of FSH preparations lacking HCG and virtually devoid of LH activity (Ubaldi et al. 1996b
) have failed to eliminate premature luteinization. Premature luteinization does not negatively affect oocyte or embryo quality (Hofmann et al., 1993a
; Legro et al., 1993
; Ubaldi et al., 1996b
; Chetkowski et al., 1997
) but may reduce the success of assisted reproduction techniques through secretory endometrial transformations that hamper embryo implantation (Shulman et al., 1996
). In the current study, as expected from the well-known interaction between LH activity and theca cell LH receptors, we found a positive correlation between the administered LH activity dose and follicular phase testosterone concentration (Figure 3
); conversely, we could not identify any significant relationship between follicular phase progesterone levels and the administered LH activity dose or follicular phase LH and HCG levels. On the other hand, we found a strong positive correlation (r = 0.447; P < 0.00005) between the administered FSH dose and follicular phase progesterone levels (Figure 3
). This finding confirms a previous report showing a similar correlation between serum FSH and progesterone levels (Ubaldi et al., 1996b
) and suggests that premature luteinization instead of being related to excessive follicular phase LH activity could be due to an increase of granulosa cell steroidogenetic function caused by intense FSH stimulation. The concept that premature luteinization is not dependent upon moderate increments in granulosa cell stimulation by LH or HCG is also supported by our finding of a lack of follicular phase progesterone levels increments caused by the daily administration of 50 IU of HCG (corresponding to about 300 IU of LH) in FSH treated patients (Filicori et al., 1999b
). Furthermore, these results also suggest that it may be possible to reduce the occurrence of premature luteinization by using gonadotrophin preparations with both LH and FSH activity, as we have reported that this combination permitted the reduction of the overall FSH dose employed in COS (Filicori et al., 1999b
, 2001
).
To summarize, we confirmed that in COS the LH activity content of menotrophins shortens treatment duration and we determined that even reduced amounts of LH activity play important roles in folliculogenesis and steroidogenesis. The HCG content of HMG appears to contribute in a relevant manner to the overall LH activity of these preparations; due to its longer half-life, HCG may be actually better suited than LH itself to provide a stable stimulation of theca and granulosa cell LH receptors. Contrary to a widely held belief, we found that FSH rather than LH or HCG is responsible for premature follicle luteinization during gonadotrophin administration. Thus, follicular phase LH activity supplementation is unlikely to cause premature luteinization or embryo implantation disorders. Finally, the findings of this study once again (Filicori et al., 2001, 2002
) indicate that LH and/or HCG administration can be employed selectively to curtail the development of small non-reproductively competent ovarian follicles. Further studies will be needed to determine if selective LH and/HCG administration could be applied to reduce the risk of ovulation induction complications such as OHSS.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 2, 2002; accepted on April 9, 2002.