1 The Fertility Clinic, Skive Sygehus, DK-7800, Skive and 2 Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen, Copenhagen, Denmark
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Abstract |
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Key words: GnRH agonist/IVF/luteinizing hormone/ovarian response/pituitary suppression
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Introduction |
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The aim of this prospective study was to evaluate further ovarian response and pregnancy outcome in relation to mid-follicular LH levels after down-regulation with GnRH agonist and ovarian stimulation with rFSH and at the same time to approach the mechanisms, determining the residual LH activity in these women.
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Materials and methods |
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Blood samples and hormone assays
Blood samples were taken on stimulation days 1 and 8. Sera were immediately analysed for estradiol and aliquots frozen at 20°C for subsequent analysis of LH. LH was measured by time-resolved immunofluorometric assay, the AutoDelfia® hLH spec. kit (Wallac Oy, Turku, Finland). The assay was performed at the Department of Clinical Biochemistry, Odense University Hospital, Odense, Denmark in order to compare data with those of Westergaard, who used the same department and the same assay (Westergaard et al., 2000). The detection limit of the LH assay as given by the manufacturer is 0.05 IU/l and Department of Clinical Biochemistry, Odense found the intra- and interassay variation of samples containing 0.10.2 IU/l LH to be <3% (Westergaard et al., 2000
). Estradiol was monitored in our laboratory using a Vidas® kit (bioMerieux, France) showing a detection limit of 33 pmol/l.
Statistical methods
Statistical differences were evaluated using analysis of variance (ANOVA), 2 test or linear regression analysis as appropriate.
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Results |
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Patient characteristics for all patients as a whole, and divided into the four groups according to the LH levels of day 8, are shown in Table I. There were no differences among the groups regarding infertility diagnosis and number of previous IVF/ICSI attempts, and pre-treatment levels of FSH did not differ significantly (ANOVA, P > 0.10, not significant). Age and BMI did not differ among the groups (Table II
).
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The number of retrieved oocytes gradually increased from 8.7 ± 1.0 in the group with LH levels <0.5 IU/l to 10.4 ± 0.9 in the group with LH levels >1.51 IU/l, however, this difference did not reach statistical significance (Table III). The number of fertilized oocytes and the frequency of fertilization were significantly higher in the two middle groups as compared with the two flanking groups. The number of positive pregnancy tests was similar in the <0.5, 0.511.0 and in the 1.011.5 IU/l group, whereas the number was significantly reduced in the >1.51 IU/l group compared with the two middle groups. The number of clinical pregnancies was significantly lower in the >1.5 IU/l group compared with the 0.511.0 IU/l group, whereas the 1.011.5 IU/l group reached a level of significance between 0.05 and 0.1. The implantation rate of embryos from the <0.5 IU/l group was significantly increased compared with the >1.51 IU/l group (P < 0.05), whereas the implantation rate of embryos from the two middle groups did not differ significantly from the two flanking groups. Similar pregnancy rates were observed when the median LH level on day 8 (i.e. 0.86 IU/l) was used to create a low and a high LH group (data not shown).
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When the median number of oocytes retrieved (i.e. 9.0 oocytes) was used to create two groups with a high and low number of oocytes, pregnancy rates in these two groups were similar (45.3 versus 52.0%).
The pregnancy outcome correlated to the level of LH on day 8 is shown in Table IV. No statistical difference was observed in relation to early pregnancy loss and the level of LH on day 8.
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Discussion |
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Residual levels of LH on stimulation on day 8 were found to have a significant impact on the ovarian response in GnRH agonist treated women receiving rFSH. Estradiol production on day 8 and levels of estradiol on day 8 per oocyte retrieved correlated positively to LH levels on day 8 suggesting that follicles exposed to the highest levels of LH were the most steroidogenically active on day 8. This supports other findings (Phelps et al., 1999; Filicori et al., 1999
); in the latter study GnRH agonist down-regulated women receiving exogenous HCG in combination with rFSH developed a stronger estradiol response compared with those who did not receive HCG, suggesting that availability of androgen substrate was a major determinant for estradiol output in follicles approaching ovulation. The present data are also in accordance with a recent preliminary report (Fanchin et al., 2001
), which defined two groups based on LH measurements on days 6 and 9 (LH levels being consistently higher or lower than 2 IU/l). Higher peak estradiol concentrations were found in the high LH group compared with the low LH group. Other studies have shown that exogenous administered LH activity, either in the form of LH or HCG, has a positive effect on the estradiol output (The European Recombinant Human LH Study Group, 1998
; Westergaard et al., 2001
).
Consumption of FSH also showed a significant inverse relationship with LH levels on day 8 resulting in a reduction of 28% when comparing the low LH group (i.e. <0.5 IU/l) with the high LH group (i.e. >1.51 IU/l). The circulating LH in the present study was derived from endogenous stores, but in combination with the results of others (Filicori et al., 1999, 2001
), who used exogenous gonadotrophins, this suggests that LH plays a significant role in the development of pre-ovulatory follicles. We also found a significant shortening of the duration of ovarian stimulation and an acceleration of follicle development, in accordance with other observations (Filicori et al., 1999
, 2001
; Fanchin et al., 2001
). This is in contrast to Westergaard, who found that the duration of ovarian stimulation was unrelated to the administration of exogenous LH and HCG (Westergaard et al., 2001
).
Although the actual number of oocytes retrieved did not differ significantly among the four groups, the quality of the retrieved oocytes seemed to be affected by the LH levels on day 8. The number and the frequency by which they underwent fertilization was significantly correlated to LH levels; the two middle groups (i.e. LH levels between 0.511.0 and 1.011.5 IU/l) perform significantly better than the two flanking groups. This contrasts with the findings of Fanchin et al. (2001), who found significantly higher numbers of oocytes, and significantly higher numbers of embryos plus a significantly higher pregnancy rate in the group of women with LH measurements >2 IU/l on both days 6 and 9 compared with the group of women in which both LH measurements were <2 IU/l. The study by Fleming also found a significantly increased number of fertilized oocytes and supernumerary embryos per case in the group of women with mid-follicular LH above 0.5 IU/l compared with those below (Fleming et al., 1998). This difference is not readily explainable, but may relate to the different mode of pituitary down-regulation or to the definitions of more groups in the present study.
Taken together, there are now substantial data to suggest that LH activity, derived from either endogenous stores or administered exogenously, in GnRH agonist treated women has a significant impact on the ovarian response during ovarian stimulation.
The present study was unable to demonstrate differences in pregnancy outcome when median LH levels on day 8 was used to create two groups. However, dividing data into four groups, we found that LH levels between 0.51 and 1.5 IU/l (i.e. the two middle groups) exhibit the best chances of a pregnancy, whereas LH levels >1.5 IU/l and <0.5 IU/l show a reduced frequency (i.e. the two flanking groups). This suggests that the two middle groups experience sufficient LH to sustain pregnancy. The low LH group tended to show a reduced pregnancy rate, but this group represented too few cases to draw any conclusions regarding pregnancy outcome and early pregnancy loss. However, the frequency of early pregnancy loss did not seem to correlate to the LH levels on day 8. It is remarkable that pregnancy outcome is significantly reduced in the group of women with LH day 8 >1.5 IU/l compared with the 0.511.0 group. This is in contrast to other studies which found superior pregnancy rates in the high LH group when the median LH level is used to create two groups (Phelps et al., 1999; Fanchin et al., 2001
; Westergaard et al., 2001
). In addition, this group is relatively small and other studies are needed to confirm the findings. However, the results may suggest that a relatively low `ceiling level' of LH actually does exist; or perhaps more likely, that this group of women experiences other endocrinologically related fertility problems.
The present study uses a single measurement of LH on day 8 to correlate ovarian response and pregnancy outcome. A more detailed LH monitoring during the day and over time could provide more detailed information, since it cannot be excluded that short-term, intra-patient variations of LH secretion occur and that LH levels may vary during FSH treatment. However, the significant correlation between estradiol and LH levels day 8 suggests that a single measurement in fact provides a good measure for the residual LH activity present during the follicular phase. Further, it should be emphasized that the present study is unable to show whether LH is the mediator of outcome or a marker. An important question for future studies to address should therefore be the exact role of LH in affecting outcome.
In conclusion, the present study suggests for the first time that the regime and the dose of GnRH agonist in connection with ovarian stimulation have an important effect on the residual LH activity in circulation. In addition, the concentrations of LH during the course of ovarian stimulation should remain in the middle range and neither become too low nor too high in order to secure the best chance for a successful treatment.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 15, 2001; resubmitted on February 20, 2002; accepted on May 2, 2002.