Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong Special Administrative Region, Peoples Republic of China
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Abstract |
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Key words: clomiphene citrate/endometrial receptivity/follicular vascularity/power Doppler
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Introduction |
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Because of its antiestrogenic action on the reproductive tract, poor quality cervical mucus (Gelety and Buyalos, 1993) and delayed endometrial maturation (Massai et al., 1993
) have been documented in patients exposed to clomiphene citrate. These adverse effects are augmented by the relatively long half-life (
5 days) of clomiphene citrate, which is usually given daily from day 5 to day 9 of the menstrual cycle. Therefore, these negative effects may be extended into the peri-implantation period and reduce the pregnancy rate. Recently, Marinko et al. reported more rapid follicular growth and higher pregnancy rates following intrauterine insemination in those patients who started clomiphene citrate on day 1 of the cycle rather than on day 5 (Marinko et al., 1999
). The authors suggested that a longer period without clomiphene citrate might enable the rising estradiol to replace clomiphene citrate from peripheral receptors and possibly create a uterine environment that was more conducive to pregnancy. It remains unknown whether the timing of clomiphene citrate administration has any effect on oocyte quality and endometrial receptivity, which may affect the pregnancy rate.
The purpose of this randomized, double-blind study was to compare the perifollicular vascularity and endometrial receptivity of ovulatory women who started clomiphene citrate on day 2 and day 5.
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Materials and methods |
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Basal serum FSH and LH levels were taken on the second day of a natural cycle. They started to attend the clinic at around 08:00 from 18 days before the next expected period for regular transvaginal scanning and hormonal assays including serum LH and estradiol (E2). The assessment was repeated every 12 days and then daily when the diameter of the leading follicle was 16 mm until the day of LH surge. The LH surge was taken when the LH level was >20 IU/l and double the mean of the LH levels over the previous 3 days. The women were seen again 7 days after the LH surge for ultrasound assessment and serum E2 and progesterone measurement.
All ultrasound examinations were carried out by W.C. using a 6.5 MHz vaginal probe (Aloka, Model SSD-5500; Aloka Co. Ltd, Tokyo, Japan) at around 09:00 after the women had emptied the bladder. During scanning, the number/size of follicles and the endometrial thickness were recorded. The vascularity of each follicle was subjectively graded using Power Doppler imaging according to a published grading system (Chui et al., 1997), i.e. grade 1:
25% of the circumference; grade 2: 2650%; grade 3: 5175%; grade 4: >75%. Grades 1 and 2 were considered as low-grade vascularity whereas grades 3 and 4 were high grade.
Doppler flow parameters of uterine and subendometrial vessels were then measured. Flow velocity waveforms were obtained from the ascending main branch of the uterine artery on the right and left side of the cervix in a longitudinal plane before it entered the uterus. The gate of the Doppler was positioned when the vessel with good colour signals was identified on the screen. The pulsatility index (PI) and resistance index (RI) of the uterine arteries were calculated electronically when three similar, consecutive waveforms of good quality were obtained. As no significant differences in the Doppler velocimetry indices between the left- and right-sided uterine vessels were obtained, the data were combined and the average value of both vessels was used. The uterus was visualized in the sagittal plane to include the whole endometrium and a colour flow map was superimposed on the upper two-thirds of the endometrium. Doppler flow indices of vessels seen at the subendometrial region were studied at random and the lowest values for resistance to flow were recorded. The intra-observer coefficient of variation was 9.6% for PI and 4.1% for RI.
Assignment
Women were then monitored in a stimulated cycle during which they were randomized to receive 50 mg of clomiphene citrate (Clomid; Merrell, Staines, Middlesex, UK) daily for 5 days from either day 2 or day 5 of the menstrual cycle. The randomization process was done by computer-generated random numbers put in sealed opaque envelopes. In group A, 50 mg of clomiphene citrate was taken daily from day 2 to day 6 and the placebo was taken from day 5 to day 9, while in group B the placebo was taken from day 2 to day 6 and 50 mg of clomiphene citrate was taken from day 5 to day 9. The above assessment performed during a natural cycle was repeated.
Serum E2 was measured using a commercially available radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA, USA) and serum FSH, LH and progesterone were measured using commercially available kits (Chiron Diagnostics Corporation, East Walpole, MA, USA). The intraobserver variation was not statistically significant.
Masking
Both the women and the doctors were blinded to clomiphene citrate and placebo, which were packaged by the hospital pharmacist and had identical appearance. The codes for the drugs were revealed only after the completion of the study and statistical analysis.
Statistical analysis
The primary outcome measures were grading of the perifolliclar vascularity, endometrial thickness and uterine arteries pulsatility and resistance index. Assuming that 50% of patients taking clomiphene citrate from day 2 to day 6 and 5% of patients taking clomiphene citrate from day 5 to day 9 had high grade perifollicular vacularity, the sample size required would be 15 in each arm to give a test of significance of 0.05 and a power of 0.8 (Sigmastat; Jandel Scientific, San Rafael, CA, USA). As continuous data were not normally distributed, results were given as median (range). Statistical tests were carried out by MannWhitney U-test, Fishers exact test and 2-test, where appropriate. Two-tailed P < 0.05 was taken as significant.
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Results |
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No significant differences were found between the two groups with regard to the age of the women, the type and duration of infertility, body mass index and basal LH:FSH ratio (Table I). There were no differences between the two groups, after taking clomiphene citrate, in hormonal profile, follicular development, number of patients with high grade vascularity, endometrial thickness, Doppler flow indices of uterine and subendometrial vessels on the day of LH surge and 7 days after LH surge (Tables II and III
). During the natural cycles, the above parameters were also comparable in both groups (Tables II and III
).
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Discussion |
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Power Doppler imaging is a relatively new mode of Doppler ultrasonography which is more sensitive than conventional colour Doppler imaging at detecting low velocity and hence improves the visualization of small vessels. Power Doppler ultrasound analysis of individual follicles during IVF treatment has been convincingly shown to provide important information about the developmental competence and implantation potential of the corresponding oocyte (Chui et al., 1997; Bhal et al., 1999
; Huey et al., 1999
; Borini et al., 2001
). Significantly higher fertilization rates and pregnancy rates were demonstrated when oocytes were obtained from follicles with higher grade perifollicular vascularity. Similar results were also demonstrated in stimulated intrauterine insemination cycles (Bhal et al., 2001
). In this randomized study, the number of follicles
16 mm in diameter, the highest grading of perifollicular vascularity and the number of patients with high grade perifollicular vascularity were similar in those groups. These results suggested that there was no difference in the oocyte quality as graded by the perifollicular vascularity irrespective of the day of clomiphene citrate initiation. It is important to point out that this finding is presumptive as oocyte quality was not examined directly in this study.
Doppler examination of the uterine artery is a non-invasive assessment of endometrial receptivity. During IVF treatment, the implantation rate is decreased when uterine artery pulsatility index is 3.33.5 at the time of HCG administration, oocyte retrieval or embryo transfer (Dickey, 1997
). Doppler examination of the subendometrial artery is another tool with which to evaluate the endometrial receptivity (Zaidi et al., 1995
; Tohma et al., 1997
; Basir et al., 2001
). There were no significant differences between the two groups in hormonal profile, endometrial thickness and Doppler flow indices of uterine and subendometrial vessels on the day of LH surge and in the luteal phase. Again, there was no evidence to show any change in the endometrial receptivity when clomiphene citrate is started on different days of the menstrual cycle. Implantation markers in the endometrium such as integrin molecules have not been examined in this study. The only advantage of starting clomiphene citrate on day 2 over day 5 seems to be that follicular recruitment and further growth occur earlier, as indicated by the day of LH surge.
In summary, the oocyte quality graded by the perifollicular vascularity and the endometrial receptivity assessed by the endometrial thickness and Doppler flow indices of uterine and subendometrial vessels remains unchanged when clomiphene citrate is started on day 2 of the menstrual cycle rather than on day 5 in regularly ovulating women.
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Acknowledgements |
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Notes |
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References |
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Submitted on May 5, 2002; accepted on July 24, 2002.