1 Division of Reproductive Medicine, Department of Obstetrics and Gynaecology and 2 Department of Public Health, Erasmus University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
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Abstract |
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Key words: endocrine profiles/GnRHa/IVF protocols
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Introduction |
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Several authors have shown extremely low endogenous LH concentrations until 1014 days after discontinuation of the GnRHa (Smitz et al., 1992a; Donderwinkel et al., 1993
; Sungurtekin and Jansen, 1995
). It may therefore be postulated that GnRHa could be stopped earlier in the stimulation cycle, allowing pituitary recovery to occur during the luteal phase providing endogenous support of the corpus luteum. Preliminary observations indeed suggest that no premature rises in LH and progesterone concentrations took place in patients in which GnRHa was stopped earlier (Pantos et al., 1994
). The objective of the present prospective randomized controlled study was to assess whether early follicular phase cessation of GnRHa still avoids a premature rise in serum LH and to study luteal phase LH and progesterone concentrations without exogenous support of the corpus luteum.
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Materials and methods |
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Forty paid volunteers aged 2034 years with a normal regular menstrual cycle (i.e. 2630 days), normal body weight (body mass index 1924 kg/m2) and no history of infertility or any endocrine abnormalities served as controls. Daily blood sampling and transvaginal ultrasound was performed, as published previously (Schipper et al., 1998).
Study protocol
All patients were treated with the so called long protocol. The GnRHa Decapeptyl® (Ferring Nederland B.V., Hoofddorp, The Netherlands) 0.1 mg s.c. daily injections were initiated on cycle day 1. Patients were randomized on the same day (i.e. day 1 of the treatment cycle) by means of sealed envelopes for one of the three treatment groups A, B or C (20 patients each). Down-regulation was confirmed (reflected by serum oestradiol concentration <150 pmol/l) after 3 weeks GnRHa use, and ovarian stimulation was initiated using human menopausal gonadotrophin (HMG) (Humegon®; N.V. Organon, Oss, The Netherlands) 3 amp/day (=225 IU) i.m. Ultrasound examination was performed from stimulation day 6 onwards every other day until the leading follicle reached a diameter of at least 15 mm. From that day onwards ultrasound examinations were performed daily. Blood samples were drawn on the first day of GnRHa, on the first day of administration of HMG, on the third day of HMG, on each day the patient visited the outpatient clinic for ovarian response monitoring, on the day of human chorionic gonadotrophin (HCG), on the day of oocyte retrieval, and every other day thereafter. Rapid serum oestradiol measurements were performed on day 21 (after 3 weeks use of GnRHa) and following every ultrasound examination. HCG (Pregnyl®; N.V. Organon) 10 000 IU was administered i.m. as a single bolus on the day the diameter of the leading follicle was at least 18 mm and at least 3 follicles >10 mm were present. Oocyte retrieval was performed 35 h later and blood samples were drawn immediately prior to this. Embryos were transferred after 4 days of culture.
Patients in group A received GnRHa until the day of HCG, and subsequent luteal support; HCG 1500 IU i.m. on the day of oocyte retrieval and 2, 4 and 6 days thereafter (conventional long protocol). Patients in group B stopped GnRHa on the third day of HMG stimulation and received no luteal support (see Figure 1). Group C used GnRHa until the day of HCG and received no luteal support. To diminish the risk of ovarian hyperstimulation syndrome (OHSS), only patients with oestradiol concentrations below 8000 pmol/l on the day of HCG received luteal support with HCG in group A. In case oestradiol concentrations were above 8000 pmol/l, micronized progesterone (600 mg/day intravaginally) was given and patients were excluded from further analysis. The oestradiol threshold of 8000 pmol/l was arbitrarily chosen. This was based on our own unpublished observations. It has previously been suggested that when a patient is considered to be at particular risk of developing OHSS, it is recommended that progesterone rather than HCG should be used for luteal support (Akande et al., 1996
). However, no absolute threshold for oestradiol could be found to predict an OHSS risk (Mathur et al., 1996
).
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Statistical analysis
Potential differences in patients' ages and oestradiol concentrations on the day of HCG were tested using the KruskalWallis test. To determine differences in luteal phase hormone profiles the mean area under the curve (AUC) was calculated for LH, FSH, and progesterone during the luteal phase. P values below 0.05 were considered to indicate significant differences. Statistical analysis was performed using commercially available software packages (GraphPad prism; Statistical Package for Social Sciences, SPSS, SPSS inc. Chicago, IL, USA).
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Results |
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LH and FSH concentrations are depicted in Figure 2. Median LH concentration on the day of HCG were 0.6 IU/l (range <0.091.5), 0.4 IU/l (range <0.092.5) and 0.7 IU/l (range 0.41.0) for groups A, B and C respectively [P = 0.37 not significant (NS)]. In the control group median LH concentration on the day before LH surge was 4.8 IU/l (range 1.310.4). No premature LH rises (defined as LH concentrations above 5 IU/l) could be observed in group B patients who stopped GnRHa earlier. The median duration between GnRHa cessation and day of HCG was 7 days (range 410). Extremely low LH concentration were found in the luteal phase in groups A and C (most concentrations below assay sensitivity). The mean area under the curve (AUC) in group B (4.8) was significantly higher compared to groups A or C (0.4, and 0.3 respectively) (P = 0.01 and 0.02 respectively) but significantly lower (P < 0.001) versus controls (39.5). Median FSH concentrations on the day of HCG were 6.9 IU/l (range 5.610.4), 6.0 IU/l (range 3.910.2), 7.2 IU/l (range 4.810.8), and 3.8 IU/l (range 1.86.3) for groups A, B, C, and controls respectively. Compared to the control group, luteal phase FSH concentrations were significantly lower in group A (P = 0.001) but similar for groups B and C. In groups B and C (and in controls) a rise in serum FSH concentrations was observed from day 10 following HCG onwards. This rise was absent in group A. The median rise in FSH concentrations between day 10 and 14 was 0.0, 2.6, 2.6 and 0.5 IU/l for groups A, B, C and controls respectively. This was significantly different (P = 0.005) between groups.
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Discussion |
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If impaired luteal progesterone production were caused by prolonged pituitary suppression after GnRHa use, this may normalize if pituitary recovery took place earlier in the luteal phase. In the present prospective, randomized study, it was investigated whether pituitary recovery and endogenous corpus luteum support would occur if GnRHa were stopped early during ovarian stimulation. The entire follicular and luteal phase was studied and normo-ovulatory women served as controls. Follicular-phase characteristics were not different in the three different treatment groups (except for a minor unexplained difference in oestradiol concentrations). Hence, cessation of GnRHa early in the follicular phase did not affect ovarian stimulation by exogenous gonadotrophins. LH or progesterone rises were not observed in any patients prior to HCG. These results confirm previously published observations (Pantos et al., 1994). After continuation of GnRHa until HCG, LH concentrations were extremely low during the subsequent luteal phase both with or without luteal support. Several authors also found early and late luteal LH serum concentrations below 1 IU/l after conventional GnRHa use until HCG (Smitz et al., 1988
; Urbancek et al., 1990
; Valbuena et al., 1997
). LH concentrations remain extremely low for at least 14 days after discontinuation of GnRHa (Donderwinkel et al., 1993
). This was confirmed in the present study. However, the present study shows for the first time that after stopping GnRHa earlier in the follicular phase, LH concentrations in the late luteal phase partially recover. Smitz et al. found an earlier increase in LH concentration in the day of HCG after early cessation of GnRHa but clearly no LH rise or progesterone rise (Smitz et al., 1992b
). In the present study, from day 8 after HCG, a slight increase in LH concentrations was found, but these levels did not reach concentrations as measured in regularly cycling controls. In the patients who stopped GnRHa on stimulation day 3 the interval between cessation of GnRHa and HCG was 713 days. Hence, the observation period from the day of discontinuation of the GnRHa until the 12th day after oocyte retrieval varied from 16 to 22 days in this group. It can be concluded that some pituitary recovery occurs 1622 days after GnRHa cessation. However, LH concentrations were still below the physiological range (<0.091.9 IU/l). Compared to regular cycling controls, in all three study groups more corpora lutea were present to produce steroids in the luteal phase. The higher concentrations of oestradiol and progesterone itself could cause extremely low LH concentrations in the luteal phase by a strong negative feedback mechanism (Gibson et al., 1991
). FSH concentrations rose in the late luteal phase in both groups without luteal support. This rise may have been secondary to reduced negative feedback associated with decreased progesterone concentrations.
Progesterone production occurred in both groups without luteal support. However, distinctly lower concentrations were reached and an earlier decrease in progesterone production was noted compared to the group with luteal support. This latter observation is in agreement with previous reports (Smitz et al., 1987; Valbuena et al., 1997
). Surprisingly, no difference in progesterone production was found comparing shorter or longer GnRHa use, despite the partial recovery of LH concentrations when GnRHa was stopped earlier. This may be due to a discrepancy between bioactive and immunoassayable LH following GnRHa use (Meldrum et al., 1984
) or irreversible luteolysis before the onset of stimulation by endogenous LH. It seems likely that during the early luteal phase the corpus luteum is supported by the HCG bolus (10 000 IU) administered 35 h before oocyte retrieval. This effect lasted for approximately 1 week as can be seen from the relationship between decreasing HCG concentrations and the duration of progesterone production (Figure 5
). In patients receiving luteal support by HCG, progesterone concentrations reached a higher maximum as compared to patients without luteal support or normal regularly cycling controls. Furthermore, the increase in progesterone concentrations lasted longer while the decrease started later. This was probably due to higher HCG concentrations in the luteal phase due to HCG supplementation. Progesterone concentrations decreased the moment that HCG levels fell below approximately 30 IU/l in all three groups.
In natural cycles luteal progesterone concentrations increase to a maximum of 70 nmol/l. In all groups, concentrations substantially higher than these were found. The effects of high progesterone concentrations on implantation chances in stimulated cycles are unclear (Pellicer et al., 1996). Since pregnancies occurred in all three groups, it seems fair to conclude that the described hormonal features did not preclude implantation. Due to the small sample size, the possibility that implantation chances are impaired cannot be excluded. Withholding luteal support resulted in an earlier decrease in progesterone concentrations. Effects on implantation chances remain unclear. Progesterone concentrations in the control group were at a more steady level. It is unknown whether a sharp decrease in progesterone concentration could be detrimental despite preceding supraphysiological progesterone levels.
In conclusion, this study shows that after cessation of GnRHa earlier in the follicular phase, a premature rise in LH or progesterone concentration is still prevented. In addition, it was found that after earlier cessation of GnRHa, luteal immunoassayable LH concentrations recovered partially. However, no effect on luteal progesterone production could be observed. Progesterone production in IVF patients without luteal support was higher as compared to the natural cycle, but lower compared to patients with luteal support. As progesterone profiles in the luteal phase were not different after earlier discontinuation of GnRHa compared to continuation until HCG, it is concluded that endogenous support of corpus luteum function in the second half of the luteal phase remains insufficient. IVF treatment without luteal support might become a reality when GnRH antagonists become available in the near future. However, other factors potentially involved in impaired luteal phase gonadotrophin secretion such as the preceding bolus injection of HCG or supraphysiological luteal phase steroid feedback should also be considered.
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Acknowledgments |
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Notes |
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* Presented during the 14th Annual ESHRE Meeting, Göteborg, Sweden, 1998
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References |
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Submitted on June 9, 1999; accepted on September 17, 1999.