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Department of Obstetrics and Gynecology, University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China
Address all correspondence and requests for reprints to: Dr. Carina C. W. Chan, Department of Obstetrics and Gynecology, University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong SAR, China. E-mail: cwcchan{at}graduate.hku.hk.
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Abstract |
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Objectives: The objective of this study was to compare the effectiveness of 250 and 500 µg recombinant hCG (r-hCG), which represented the lower and upper limits of the dose range, in inducing final oocyte maturation during IVF and intracytoplasmic sperm injection cycles.
Design: This was a prospective, randomized, double-blind study.
Setting: This study was performed at an IVF clinic in a teaching hospital.
Patients: Sixty patients with an indication for intracytoplasmic sperm injection were studied.
Intervention: The treatment dose used was 250 or 500 µg r-hCG.
Main Outcome Measures: The percentage of metaphase II oocytes retrieved per patient, as an indicator of oocyte maturation, and the hormone profiles of the treatment cycle starting from the day of hCG up to hCG+10 d were the main outcome measures.
Results: The percentage of metaphase II oocytes was similar in the two groups (89.3% vs. 86.0%; P = 0.326) despite higher serum and follicular fluid hCG levels on hCG+2 and hCG+4 d, as was the follicular fluid to serum hCG ratio in the 500-µg r-hCG group. Serum estradiol and progesterone levels were comparable initially, but became significantly higher in the 500-µg r-hCG group on hCG+10 d.
Conclusion: The two doses of r-hCG were equally effective in inducing final oocyte maturation. It remains unclear whether the higher midluteal estradiol and progesterone levels in the 500-µg r-hCG group confer any benefit.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Despite the use of u-hCG, there are situations in which no oocyte can be retrieved even after administration of a high dose of u-hCG. Apart from the different individual thresholds of serum hCG required to stimulate final oocyte maturation, the other commonly cited reason to explain the empty follicle syndrome is the failure to provide biologically active hCG, due to manufacture, packing, or shelf life of the drug (3). Recombinant hCG (r-hCG) offers remarkable batch to batch consistency, high purity, and high specific activity (4, 5). A pharmacokinetic study of u-hCG and r-hCG showed that the mean serum profile after administration of 250 µg r-hCG was similar to that seen after the administration of 5000 IU u-hCG (6). Clinical studies have confirmed that treatment with 250 µg r-hCG leads to equivalent ovulation rates as 5000 IU u-hCG in anovulatory women undergoing ovulation induction (7). r-hCG at a dose of 250 µg has also been shown to be similar to 5000 IU u-hCG in the number of oocytes retrieved per patient in IVF cycles (8, 9). When a higher dose of r-hCG (500 µg) was compared with 250 µg r-hCG, the mean number of oocytes retrieved was the same, but the numbers of two pronuclear fertilized oocytes and cleaved embryos were significantly higher with 500 µg r-hCG (10). The luteal phase serum progesterone (P) and hCG concentrations were also higher in patients receiving the higher dose of r-hCG (10).
The primary outcome measure in these three studies (7, 8, 10) was the number of oocytes retrieved per patient as a function of follicular maturation. However, not all retrieved oocytes were mature. The proportion of MII oocytes may better reflect the effect of hCG on follicular maturation, because this indicates the resumption of meiosis. In this study we compared the effects of 250 and 500 µg r-hCG, which represented the lower and upper limits of the dose range, on the percentage of MII oocytes retrieved per patient during IVF and intracytoplasmic sperm injection (ICSI)/embryo transfer (ET) cycles as the primary outcome measure, with the hypothesis that the effects of the two different doses of r-hCG were similar. As a second objective, the hormone profiles of the treatment cycle starting from the day of hCG up to hCG+10 d were compared.
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Patients and Methods |
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Infertile patients between 18–40 yr of age who were attending the Assisted Reproduction Unit at Department of Obstetrics and Gynecology, University of Hong Kong, for ICSI/ET treatment between August 2003 and March 2004 were recruited on the day of hCG administration. Inclusion criteria were 1) body mass index less than 28 kg/m2, 2) early follicular phase serum FSH level of 10 IU/liter or less, and 3) ICSI indicated because of severe semen abnormalities (<100,000 motile spermatozoa recovered after sperm preparation) or surgically retrieved spermatozoa from epididymis or testis in cases of azoospermia. Patients were excluded if they had 1) a previous history of excessive or poor ovarian responses; 2) an excessive ovarian response, as defined by the presence of more than 15 follicles with an average diameter of 16 mm or greater; 3) a poor ovarian response, as defined by the presence of less than three follicles with an average diameter of 16 mm or greater; and 4) an indication for ICSI due to fertilization failure or less than 30% fertilization rate in previous IVF cycles. All patients gave written informed consent before participating in the study, which was approved by the joint institutional review board of University of Hong Kong and the Hospital Authority. Each patient was recruited to join the study only once.
IVF protocol
The details of the long protocol of ovarian stimulation regimen, gamete handling, ICSI, and assessment of oocyte/embryo quality at our center have been previously published (11, 12). In brief, pituitary down-regulation was achieved with a short-acting GnRH analog (Suprecur, Hoechst, Frankfurt, Germany) starting from the luteal phase of the cycle preceding the treatment cycle. Ovarian stimulation was started in the early follicular phase if pituitary suppression was complete, as assessed by a basal serum estradiol (E2) level less than 220 pmol/liter and the absence of ovarian cysts on ultrasound scan. Human menopausal gonadotropin (Pergonal, Serono, Geneva, Switzerland) was given by im injection (300 IU daily for the first 2 d, followed by 150 IU daily). The ovarian response was monitored by ultrasound scan. r-hCG (Ovidrel, Serono, Bari, Italy) was given if the leading follicle was 18 mm in diameter or larger and there were at least three follicles more than 15 mm in diameter.
Patients were randomly allocated into either the 250 or 500 µg r-hCG group according to a randomization table, with the allocation group presealed in opaque envelops by a research nurse. The sequence of allocation was concealed until interventions were assigned by the research nurse after the patients were recruited by an attending doctor. The drug was administered by a duty nurse who was not involved in the study. The clinicians and laboratory staff involved as well as the patient herself were blinded as to the dosage of r-hCG used. Transvaginal ultrasound-guided oocyte retrieval (TUGOR) was scheduled 36 h after the r-hCG injection. The local reaction to the injection sites was assessed on the day of TUGOR. Any local reaction, including itchiness, redness, swelling, bruising, and pain, was recorded, and severity was assessed as mild, moderate, or severe by the patients.
TUGOR was performed using a 16-gauge, double-channel needle (Cook IVF, Cook, Australia) under ultrasound guidance with a 5-MHz vaginal probe fitted with a needle guide. The double-channel needle allowed aspiration and flushing of follicles that were larger than 10 mm on both sides. Follicular fluid was collected from the first mature follicle aspirated on both sides using medium-free collection tubes. Each follicle was flushed once with culture medium, and the fluid from aspiration and flushing was examined by the embryologist (W.S.B.Y. or E.Y.L.L.). Denuded oocytes were cultured in G-1 version 3 medium for 2 h. MII oocytes were identified by the presence of a polar body. ICSI was performed on MII oocytes only. The injected oocyte was additionally cultured in a 15-µl droplet of G-1 version 3 medium under mineral oil in a CO2 incubator. Fertilization was checked 14–18 h after the injection procedure. An oocyte was considered to be normally fertilized when two pronuclei were visible. When no pronucleus or only one pronucleus was visible, the oocyte was cultured for another 3–4 h and examined again. Normally cleaving embryos were replaced in the uterine cavity 48 h after the retrieval. Immediately before ET, embryos were examined for the number and regularity of blastomeres, and the degree of fragmentation.
A maximum of two normally cleaved embryos were replaced into the uterine cavity 48 h after the retrieval. Excess good quality embryos were frozen for subsequent transfer if the woman was not pregnant in that cycle. All fresh embryos were cryopreserved if serum E2 on the day of r-hCG injection exceeded 20,000 pmol/liter to reduce the risks of ovarian hyperstimulation syndrome (OHSS), which was graded into mild, moderate, and severe degrees (13). The luteal phase was supported by P vaginal pessaries (Cyclogest, Cox Pharmaceuticals, Barnstaple, UK; 400 mg twice daily for 14 d starting from the day of ET). Blood was taken for serum E2, P, hCG, and vascular endothelial growth factor (VEGF) assays on the days of hCG, TUGOR (hCG+2 d), and ET (hCG+4 d) and 6 d after ET (hCG+10 d). A urinary pregnancy test was performed 20 d after administration of the ovulatory dose of r-hCG.
Only clinical pregnancies were considered and were defined by the presence of one or more gestational sacs or the histological confirmation of gestational product in miscarriages. Ongoing pregnancies were those pregnancies beyond 10–12 wk of gestation, at which stage the patients were referred out for antenatal care.
Serum E2, P, and hCG concentrations were measured using commercially available kits (Automated Chemiluminescence ACS-180 System, Bayer Corp., Tarrytown, NY). The sensitivity of the E2 assay was 10.0 pg/ml, and the intra- and interassay coefficients of variation were 8.1% and 8.7%, respectively. The sensitivity of the P assay was 0.11 ng/ml, and the intra- and interassay coefficients of variation for P were 5.0% and 7.8%, respectively. The sensitivity of the hCG assay was 2 IU/liter, and the intra- and interassay coefficients of variation for hCG were 1.8% and 4.9%, respectively. Serum VEGF was measured by a quantitative sandwich enzyme immunoassay technique (Quantikine, R&D Systems, Inc., Oxon, UK), which was designed to measure VEGF165 levels. The minimum detectable VEGF concentration by the assay was 9.0 pg/ml. The interassay coefficients of variation were 8.8%, 7.0%, and 6.2% at concentrations of 65, 250, and 1,003 pg/ml, respectively, whereas the intraassay coefficients of variation were 6.7%, 4.5%, and 5.1% at concentrations of 54, 235, and 910 pg/ml, respectively.
Statistical analysis
The primary outcome measure was the percentage of MII oocytes per patient. From our own statistics, the mean percentage of MII oocytes from 40 patients treated with IVF and ICSI was 87.0% with an SD of 13.8% (12). It was assumed that 250 µg r-hCG was as effective as 500 µg r-hCG in inducing oocyte maturity if the percentage of MII oocytes per patient fell within 1 SD of the original proportion (i.e. 13.8%) when the patients were treated with 500 µg r-hCG. To fulfill such an assumption, the number of subjects required in each group was 24 to give a power of 90% at the 5% significance level. Hence, 30 patients were recruited for each group to allow dropout. The serum and follicular fluid hCG levels and their ratio; the serum E2, P, hCG, and VEGF levels on various days after r-hCG administration; outcomes of the IVF cycle; and side effects were secondary outcomes measures.
Continuous variables were expressed as the mean ± SD and were compared between the two groups using Student’s t test. Categorical variables were compared using 
2 test or Fisher’s exact test where appropriate. Differences were considered significant at P < 0.05.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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. There were no differences between the two groups in terms of these prerandomization characteristics. No differences were observed in the number of follicles aspirated, the number of MII oocytes, and the percentage of MII oocytes to oocytes retrieved per patient between the two groups (Table 1).
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Graphical representation of the changes in the serum E2, P, hCG, and VEGF levels over the treatment cycle is shown in Fig. 1. Serum E2 and P levels were comparable on hCG, hCG+2, and hCG+4 d, but the levels became significantly higher in the group with 500 µg r-hCG on hCG+10 d (P = 0.039 and P < 0.001, respectively). In the 500 µg r-hCG group, the serum and follicular fluid hCG levels on hCG+2 d as well as the follicular fluid to serum hCG ratio were significantly higher (Table 1
). The serum hCG level remained higher in the 500 µg group on hCG+4 d. The difference disappeared on hCG+10 d, when the serum hCG level approached zero. The serum VEGF levels showed a consistent rise starting from the hCG day, but the levels were similar in the two groups. It appeared that the serum VEGF level on hCG+10 d was slightly higher in the 500 µg group, but this was not statistically significant (by Student’s t test, P = 0.536).
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Side effects
The side effects of the administration of r-hCG are shown in Table 3. Six patients (10%) had OHSS; in five, the OHSS was mild. One patient required hospital admission for fluid replacement. No other intervention was necessary to treat the OHSS. Local reactions at the injection site of r-hCG were uncommon, except for pain. Among the 43 patients (72%) who complained of pain from the injection site, 11 (18%) rated it as severe. However, the complaint was equally divided between the two groups.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The serum and follicular fluid hCG levels were significantly higher in the group who received a higher dose of r-hCG. The follicular fluid to serum hCG ratio was also significantly higher in the latter group. These observations, however, did not translate into a higher number of oocytes retrieved, percentage of MII oocytes, or other cycle characteristics, including pregnancy outcome, although the sample size was too small to detect a small difference in the pregnancy rate. Oocyte maturation can be achieved with hCG doses of lesser amplitude than those required for follicular rupture (18, 19, 20). In fact, a low serum hCG level before oocyte recovery (<100 IU/liter) was equally potent as the higher levels at initiating maximal oocyte maturity and achieving a similar percentage of MII oocytes recovered (21). The lowest serum hCG level on the day of hCG administration was 37 IU/liter in our study. The percentage of MII in this particular patient was 86%. Although the critical blood level of hCG capable of producing a maturational effect on the oocyte could not be determined, this level seemed adequate.
A previous study has shown that the ratio of the level of hCG in follicular fluid to that in serum was a good marker for pregnancy (22). It was suggested that a follicular fluid level of 62 IU/liter was the lowest level of hCG associated with pregnancy, and the ratio of the follicular fluid to serum hCG level must be at least 0.46 for pregnancy to occur. A later study did not find a significant difference in the follicular fluid to serum hCG levels for pregnant and nonpregnant patients, and the lowest ratio for pregnancy was 0.54 (23). Our study possessed the same limitation as the study by Stelling et al. (23), in that the sample size did not have adequate power to distinguish a small difference in pregnancy rate. However, we have shown that a ratio as low as 0.27 could achieve pregnancy.
The serum E2 and P levels were significantly higher in the group with a higher dose of r-hCG on hCG+10 d, corresponding to the midluteal phase and, hence, an indirect evidence of corpus luteal function. A higher P level with 500 µg r-hCG has also been described in an earlier study (10). Whether such a difference in the serum P level confers a more favorable luteal environment for implantation is unclear. When spontaneous cycles of normal fertile women were studied, significantly higher E2 concentrations were shown in serum (24), saliva (25), and urine (26) during the midluteal phase of pregnant cycles. Stewart et al. (24) suggested that the enhanced ovarian steroid secretion in pregnant cycles could be due to a gonadotropic stimulus such as hCG from the preimplantation embryo. In contrast to those in fertile subjects, midluteal E2 and P concentrations during spontaneous cycles were similar in pregnant and nonpregnant cycles of infertile patients (27). The explanation for this difference between fertile and infertile women is unknown. There is little recent information in humans with regard to the importance of luteal E2 during stimulated cycles in patients undergoing IVF-ET. Luteal phase serum E2 and P concentrations were not related to the outcome of IVF treatment (28, 29). In contrast, Gidley-Baird et al. (30) and Hutchinson-Williams et al. (31) found significantly higher E2 levels during the midluteal period in pregnant than in nonpregnant cycles. Different luteal supports, including no support, hCG, and P, were employed, and this may explain the different results observed in these studies. The comparable pregnancy rate found in our study did not preclude the benefit of higher E2 or P, because the study was not powered to detect this difference. The E2 level was also significantly higher in the 500 µg r-hCG group. Interestingly, the incidence of OHSS was higher in the 250 µg group, although the difference was not statistically significant. This paradoxical observation might be explained by the fact that patients at risk for OHSS were excluded from the study, and the number of patients with OHSS was small.
VEGF expression has been localized to the corpus luteum in humans (32, 33). Because VEGF has a potent angiogenic property, its presence in the corpus luteum supports its role as a regulator of angiogenesis in this tissue. We observed a continuous rising concentration in serum VEGF in the luteal phase, consistent with previous studies (34, 35). This increase is important in inducing adequate angiogenesis and development of the corpus luteum. In a small study, serum VEGF levels on d 11–14 after ET in patients who conceived after IVF were found to be higher than in those who did not (36). This could suggest an augmentation of ovarian VEGF production by hCG around the onset of intrauterine gestation, not necessarily better corpus luteal function. We could not show a difference in serum VEGF levels between the two study groups. Several studies also suggested that VEGF may play an important role in the development of OHSS, because of its vascular permeability effect (34, 36, 37, 38, 39, 40). We failed to demonstrate a difference in serum VEGF levels throughout the treatment cycle between patients who developed OHSS and those who did not (data not shown), probably because our patients were not at high risk of developing this complication, and the number of those who developed OHSS was too small.
In conclusion, we have shown that 250 µg r-hCG was as effective as 500 µg r-hCG in inducing final oocyte maturation. The serum and follicular fluid hCG levels and their ratio were higher in the group receiving 500 µg r-hCG. However, very low levels of serum and follicular fluid hCG were associated with pregnancy. The other differences were the midluteal phase serum E2 and P level in favor of the 500 µg r-hCG group. Whether this results in a better luteal environment for implantation needs additional study.
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Footnotes |
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Abbreviations: E2, Estradiol; ET, embryo transfer; hCG, human chorionic gonadotropin; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; MII, metaphase II; OHSS, ovarian hyperstimulation syndrome; P, progesterone; r-, recombinant; TUGOR, transvaginal ultrasound-guided oocyte retrieval; u-, urinary; VEGF, vascular endothelial growth factor.
Received November 3, 2004.
Accepted April 18, 2005.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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