Department of Obstetrics and Gynecology, National Hospital, University of Oslo, N-0027 Oslo, Norway
1 To whom correspondence should be addressed. Email: sverre.bjercke{at}rikshospitalet.no
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Abstract |
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Key words: assisted reproduction/early pregnancy loss/FSH/LH/ovarian stimulation
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Introduction |
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It has been suggested that down-regulation with GnRH agonist in some normogonadotrophic women may result in a profound suppression of LH concentrations, which in turn impairs adequate estradiol synthesis during FSH stimulation for IVF/ICSI (Westergaard et al., 1996; Fleming et al., 1998, 2000; Janssens et al., 2000
), fertilization rates (Westergaard et al., 1996
; Humaidan et al., 2002
), the number of clinical pregnancies (Humaidan et al., 2002
), and pregnancy outcome (Westergaard et al., 2000
). Indeed, inferior IVF outcome as a result of the use of GnRH agonist and recombinant (r)FSH have also been reported by others (Flemming et al., 1996; Esposito et al., 1998). However, other studies do suggest that the so-called resting levels of LH, as seen in women undergoing down-regulation with GnRH antagonist and stimulation with purified or rFSH preparations, are sufficient to support development and maturation of follicles and oocytes in normogonadotrophic women (Chappel and Howles, 1991
; Daya et al., 1995
; Loumaye et al., 1997
; Balasch et al., 2001
).
In the present report we examine the impact of serum LH concentrations on the first day of ovarian stimulation (stimulation day 1) on the outcome of IVF and ICSI. The specific aims are: (i) to assess the usefulness of serum LH concentration on stimulation day 1 as a predictor of ovarian response, pregnancy, and the outcome of pregnancy; and (ii) by the use of receiver operating characteristic (ROC) curve analysis, to define the best threshold value, if any, to discriminate between women with low and normal LH concentration.
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Materials and methods |
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All patients received mid-luteal phase down-regulation regimen. Pituitary function was suppressed with the GnRH agonist buserelin (Suprecur, Hoechst, Germany) by four doses daily as nasal spray (600 µg/day) or nafarelin (Synarela, Pfizer, USA) by three doses daily nasal spray (600 µg/day) given for a minimum of 14 days until a withdrawal bleeding and suppressed E2 concentrations (<0.2 nmol/l) were achieved. Concomitant stimulation with human rFSH (Gonal F; Serono, Switzerland; or Puregon; Organon, The Netherlands) was then started. The starting daily FSH dose was 75300 IU according to age, experience from previous cycles, baseline FSH concentration, infertility diagnosis, and BMI. Response to stimulation was evaluated by serial vaginal ultrasound examinations and serum E2 concentrations, and the dosage of FSH was adjusted accordingly.
On stimulation day 1, before the first injection of FSH, blood samples were drawn for analysis of E2, LH and FSH. The methods used for hormone analyses have been described in previous studies (Dale et al., 1998). In brief, serum concentrations of LH and FSH were measured using DELFIA (dissociation-enhanced Lanthanide fluoroimmunoassay) kits obtained from LKB Wallac (SF-220101; Turku, Finland). Inter- assay coefficients of variation for the individual analysis were: LH 58% and FSH 8%.
Ovulation was induced with 10 000 IU hCG (Profasi, Serono or Pregnyl, Organon) when at least one follicle had a diameter of 18 mm. Oocytes were collected after 3438 h under vaginal ultrasound guidance. Handling of gametes, fertilization, and embryo culture were according to standard IVF procedures. Briefly, Universal IVF medium (Medi-Cult, Copenhagen, Denmark) was used throughout oocyte insemination and embryo culture. In ICSI cycles only ejaculated sperm were microinjected. Embryos were scored from grade 1 (high quality) to grade 4 (poor quality) according to the size and shape of blastomeres and degree of fragmentation. Embryos were transferred on day 3, except in cases with few embryos (
2), which were transferred on day 2. As a rule, and when available, two embryos were transferred. Luteal phase support up to 14 days after oocyte retrieval consisted of daily i.m. injection of 25 mg progesterone in oil or by vaginal administration of micronized progesterone 300 mg x2 (Progestan; Organon A/S, Oss, The Netherlands).
Serum concentration of -hCG was measured on day 14 after oocyte retrieval, and in case of concentrations >20 IU/l, which indicated conception, ultrasound scans were performed at 78 weeks gestation to verify the viability of pregnancy. Early pregnancy loss was defined as a biochemical pregnancy that failed to progress beyond 12 weeks gestation. Ongoing pregnancy was defined as a pregnancy that progressed beyond 12 weeks.
Data analysis
Results are expressed as mean±SD. Continuous data were analysed with Student's t-test or the MannWhitney U-test, where appropriate. The 2-test and
2-test for linear trend were used for frequencies. P<0.05 was considered statistically significant.
The discrimination attained between two compared groups (conception versus non-conception cycles and ongoing pregnancy versus early pregnancy loss) was evaluated with the ROC analysis (Hanley and McNeil, 1982; Zweig and Campell, 1993
). ROC curves are plots of all the sensitivity and specificity pairs that are possible for all levels of a particular parameter. They are constructed by plotting the false positive rate or (1 specificity) on the x-axis and the true positive rate or sensitivity on the y-axis. The best threshold value discriminating between the two conditions is typically located at the greatest distance from the diagonal. The area under the ROC curve (AUCROC) may serve as an estimate of accuracy, i.e. the ability of a particular parameter (LH concentration on stimulation day 1 in this paper) to discriminate between two conditions (conception versus non-conception and ongoing pregnancy versus early pregnancy loss).
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Results |
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Compared to non-conception cycles, conception cycles were characterized by a lower mean age of women, shorter duration of stimulation, lower total dose of FSH during stimulation, increased number of collected and fertilized oocytes, increased mean number of transferred embryos, and higher quality of transferred embryos. Hormone concentrations on first day of stimulation were not significantly different between conception and non-conception cycles (Table I). Statistically significant differences in duration and total dose of FSH stimulation, number of collected and fertilized oocytes, and hormone concentrations of stimulation day 1 were not observed between treatment cycles leading to ongoing pregnancies compared to cycles with early pregnancy loss (Table I).
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The diagnostic accuracy of serum LH concentration on stimulation day 1 to discriminate between conception versus non-conception cycles and ongoing pregnancies versus early pregnancy losses was examined with ROC analysis, which did not reveal a statistically significant diagnostic value of LH concentration (Figure 1, Table IV). In order to examine the sensitivity of ROC analysis, the analysis was repeated in the following subsets: (i) only the first cycle of each couple; (ii) all IVF cycles; (iii) all ICSI cycles; (iv) excluding cycles with GnRH agonist down-regulation for >90 days and/or presence of PCOS; (v) combination of these criteria. Serum LH concentration on stimulation day 1 did not discriminate significantly between conception versus non-conception cycles and ongoing pregnancies versus early pregnancy losses in these analyses (Table IV).
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Discussion |
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Suggestions have been made that GnRH agonist down-regulation in some normogonadotrophic women may result in profound suppression of LH concentrations, impairing adequate E2 synthesis (Westergaard et al., 1996; Flemming et al., 1998
, 2000
; Janssens et al., 2000
), fertilization rates (Westergard et al., 1996
) or the final clinical treatment outcome by increasing the risk of early pregnancy loss (Westergaard et al., 2000
). It has also been suggested that in addition to a threshold there might exist a ceiling in LH activity (i.e. an optimal range for LH action), and that excessive LH administration might impair treatment outcome in ovulation induction and assisted reproductive treatment (Shoham, 2002
). Our results, however, show that the level of LH on stimulation day 1 did not have any implication on the clinical outcome. The requirement for LH during the follicular phase is likely to be low, because <1% of pre-ovulatory follicular theca LH receptors need to be occupied to allow normal ovarian steroidogenesis (Chappel and Howles, 1991
). The fair assisted reproductive treatment results obtained with the use of rFSH and i.n. GnRH agonist tend to support this notion (Westergaard et al., 2001
). Furthermore, patients with intermediate levels of residual endogenous LH secretion on stimulation day 8 have been found to respond optimally, whereas women with high or low LH concentrations had reduced success rates after assisted reproductive treatment (Humaidan et al., 2002
). We could find no such correlation by stratification of the LH concentrations in our study (Table III).
Women with hypogonadotrophic hypogonadism (HH) most often need addition of gonadotrophin preparations containing LH activity (hMG or rLH) to the stimulation protocol in order to restore E2 producton and to improve the reproductive outcome (Shoham et al., 1991; Kousta et al., 1996
). LH of <1.2 IU/l is suggestive of HH (European Recombinant Human LH Study Group, 1998
). Accordingly, levels of LH <1.2 IU/l on stimulation day 1 in i.n. GnRH agonist down-regulated women have been applied as an indication for supplementation of LH during the FSH stimulation phase. Even after promotion of optimal follicular development in HH by adding rLH in sufficient amounts, LH remains <1 IU/l (European Recombinant Human LH Study Group, 1998
). This further confirms that, as shown in pituitary down-regulated patients, minimal circulating levels of LH are required to initiate follicle steroidogenesis. Therefore, measurements of serum immunoactive LH levels are of limited value, if any, for identifying whether a patient has enough endogenous LH activity to respond adequately to stimulation with FSH alone (Chappel and Howles, 1991
; Loumaye et al., 1997
). Thus there may be significant differences related to how longstanding minimal LH levels may affect the folliculogenesis in HH women, in contradiction to the very short period of time with low LH levels as a result of i.n. GnRH agonist down-regulation.
Other experimental and clinical studies indicate that LH is not required for follicular growth, but exogenously administered LH probably plays a primary role in complete maturation of the follicle and oocyte competence in patients with longstanding HH (Balasch et al., 1995; Fox et al., 1997
; Cortvrindt et al., 1998
). Our results indicated that this might also be the case for women who have been treated with GnRH agonist for >90 days. As there were no differences in the consumption of FSH, the ovarian response and the embryo quality regardless of whether or not i.n. GnRH agonist were used for >90 days, the lower pregnancy rate after long-term use of GnRH agonist might be due to a reduced endometrial receptivity (Table III). Another explanation could be that the dominant diagnosis within this group was extensive endometriosis, which may be associated with a reduced implantation rate (Bernhart et al., 2002
; Kao et al., 2003
). However, our results are in contrast to those of Surrey et al. (2002)
who observed a higher ongoing pregnancy rate in endometriosis patients down-regulated for 3 months before ovarian hyperstimulation compared to those who were treated with a conventional mid-luteal down-regulation regimen. In their paper, however, the type of gonadotrophin is not stated and patients with ovarian endometriosis were excluded. Due to the limited number of patients in both studies, more studies are needed to confirm these findings.
The present study shows that even profoundly suppressed LH serum concentrations (LH <0.5 IU/l) failed to affect the clinical outcome. LH suppression probably had no significant effect on estradiol biosynthesis since serum estradiol concentrations on stimulation day 1 were not significantly different when groups with various LH concentrations were compared. In a study from our own group (Tanbo et al., 2001) it has been shown that during the time of rFSH stimulation serum LH concentration decreased by an average of 35%. Furthermore androgen levels increased during rFSH stimulation in down-regulated IVF patients despite a decline in immunoactive LH during the stimulation period. FSH therefore, probably via inhibin production, is by itself capable of stimulating thecal androgen biosynthesis.
The patients in this study received two types of GnRH agonist. One half received buserelin, the other half nafarelin. It has been claimed that these two GnRH agonists might have different suppressive effects. Dada et al. (1999) showed that buserelin needed one more week to obtain the same suppressive effect on estradiol as nafarelin. Simberg et al. (1998)
showed that serum LH was lower in patients after 2 weeks of administration of buserelin compared to nafarelin. Furthermore the latter report claimed that the use of buserelin resulted in more oocytes recovered, while the fertilization rate was higher with the use of nafarelin (Simberg et al., 1998
). No studies, however, have shown differences in implantation, miscarriage and pregnancy rate to be dependent on whether buserelin or nafarelin was administered (Lockwood et al., 1995
; Simberg et al., 1998
; Dada et al., 1999
). The subjects incorporated in the first two of these studies were few and the results from the studies partly contradictory. More investigations are needed to determine whether there are any differences in outcome of IVF/ICSI after i.n. administration of buserelin or nafarelin.
In conclusion, we found that serum LH concentration on the first day of FSH stimulation is not predictive of pregnancy and the outcome of pregnancy in women who had received long-term down-regulation with an intranasal GnRH agonist.
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References |
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Submitted on October 13, 2004; resubmitted on March 9, 2005; accepted on April 29, 2005.
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