A randomized double-blind comparison of perifollicular vascularity and endometrial receptivity in ovulatory women taking clomiphene citrate at two different times

Wai Cheung1, Ernest Hung Yu Ng and Pak Chung Ho

Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong Special Administrative Region, People’s Republic of China


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: It is still controversial whether the day of clomiphene citrate initiation has any impact on the pregnancy rate. This study aimed to compare the perifollicular vascularity and endometrial receptivity of ovulatory women who started clomiphene citrate on day 2 and day 5. METHODS: Thirty-five women with regular ovulatory cycles were first monitored in a natural cycle and then randomized by computer-generated random numbers put in sealed opaque envelopes to receive 50 mg clomiphene citrate on days 2–6 or on days 5–9. The hormonal profile, the number of dominant follicles, the grading of perifollicular vascularity, endometrial thickness and Doppler flow indices of uterine/subendometrial arteries were compared between both groups. RESULTS: All the above parameters were similar for both groups on the day of the LH surge and 7 days after the LH surge. CONCLUSIONS: There were no differences in oocyte quality graded by the perifollicular vascularity and the endometrial receptivity assessed by endometrial thickness and Doppler flow indices of uterine and subendometrial vessels when clomiphene citrate was started in regularly ovulatory women on day 2 or on day 5.

Key words: clomiphene citrate/endometrial receptivity/follicular vascularity/power Doppler


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Clomiphene citrate has been used to treat women suffering from unexplained infertility, which accounts for 28% of infertility (Hull et al., 1985Go). It has been shown that clomiphene citrate increases the number of follicles produced per cycle (Randall and Templeton, 1991Go), which may improve the chance of a fertilized embryo reaching the uterus or correct an unidentifiable ovulatory dysfunction. Although it is still a matter of controversy whether pregnancy rate can be improved in patients with unexplained infertility after taking clomiphene citrate (Royal College of Obstetricians and Gynaecologists, 1998Go), a recent Cochrane review (Hughes et al., 2000Go) of five trials indicated that clomiphene citrate was associated with an increase in pregnancy rates when compared with placebo. The odds ratio (OR) for pregnancy per patients was 2.38 [95% confidence interval (CI) 1.22–4.62] and the OR for pregnancy per cycle was 2.5 (95% CI 1.35–4.62). These studies differed in the quality of randomization as only one study was randomized in a double-blind fashion and four were cross-over studies.

Because of its antiestrogenic action on the reproductive tract, poor quality cervical mucus (Gelety and Buyalos, 1993Go) and delayed endometrial maturation (Massai et al., 1993Go) 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., 1999Go). 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Protocol
Women attending Assisted Reproduction Unit, Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital were recruited if they satisfied the following inclusion criteria: (i) age of women between 25 and 40 years at the time of screening; (ii) regular menstrual cycles of 28–30 days with mid-luteal progesterone level >30 nmol/l; (iii) bilateral tubal patency as confirmed by diagnostic laparoscopy with chromotubation; (iv) body mass index between 18 and 26 kg/m2 and (v) good physical and mental health. Those with (i) history of ovarian surgery including drilling, cystectomy or oophorectomy; (ii) infertility caused by endocrine abnormalities such as hyperprolactinaemia; (iii) ultrasound diagnosis of polycystic ovaries; (iv) chronic cardiovascular, hepatic, renal or pulmonary disease; and (v) concurrent use of drugs other than those prescribed by the investigators were excluded from the study. Eligible women were extensively counselled and gave their written consent prior to participating in the study, which was approved by the Ethics Committee, Faculty of Medicine, The University of Hong Kong.

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 1–2 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., 1997Go), i.e. grade 1: <=25% of the circumference; grade 2: 26–50%; grade 3: 51–75%; 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 Mann–Whitney U-test, Fisher’s exact test and {chi}2-test, where appropriate. Two-tailed P < 0.05 was taken as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A total of 35 women were recruited: 18 women in group A (i.e. clomiphene citrate on day 2–6) and 17 in group B (i.e. clomiphene citrate on day 5–9). One patient in group A became pregnant after taking clomiphene citrate and the hormonal parameters and ultrasound assessment in the luteal phase of that cycle were not included in the comparison.

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 IGo). 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 IIIGoGo). During the natural cycles, the above parameters were also comparable in both groups (Tables II and IIIGoGo).


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Table I. Comparison of demographic data of women
 

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Table II. Comparison of hormonal profile, perifollicular vascularity and endometrial receptivity on the day of LH surge during natural and clomiphene citrate cycles
 

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Table III. Comparison of hormonal profile and endometrial receptivity 7 days after the LH surge during natural and clomiphene citrate cycles
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
There are only two studies published in the literature addressing the effect of starting clomiphene citrate on different days of the menstrual cycle. Wu and Winkel, in a study of 414 cycles in 87 anovulatory women, randomly initiated clomiphene citrate on the 2nd, 3rd, 4th or 5th day of the menstrual cycle and could not find any significant differences between the groups in terms of anovulation rates, luteal dysfunction, and normal ovulation rates (Wu and Winkel, 1989). There were no differences in pregnancy rate and pregnancy outcomes. On the other hand, Marinko et al. recently found that clomiphene citrate commenced on day 1 of the menstrual cycle, rather than day 5, resulted in more rapid follicular growth and higher pregnancy rates (25 versus 0% pregnancy rate per cycle started, P = 0.04) when intrauterine insemination was performed (Marinko et al., 1999Go). They concluded that the administration of clomiphene citrate earlier in the menstrual cycle, enabled the rising E2 to replace clomiphene citrate from peripheral receptors and possibly created a uterine environment that was more conducive to pregnancy. Moreover, the vascular resistance of the uterine artery was declining in the group who received clomiphene citrate early. It remains unclear whether different initiation day of clomiphene citrate has any effect on oocyte quality and endometrial receptivity.

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., 1997Go; Bhal et al., 1999Go; Huey et al., 1999Go; Borini et al., 2001Go). 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., 2001Go). 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 >=3.3–3.5 at the time of HCG administration, oocyte retrieval or embryo transfer (Dickey, 1997Go). Doppler examination of the subendometrial artery is another tool with which to evaluate the endometrial receptivity (Zaidi et al., 1995Go; Tohma et al., 1997Go; Basir et al., 2001Go). 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank the Department of Pharmacy, Queen Mary Hospital for preparing the packages of drugs and placebo.


    Notes
 
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, United Christian Hospital,130 Hip Wo Street, Kwun Tong, Hong Kong. E-mail: w8382982{at}netvigator.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Basir, G.S., Lam, T.P.W., Chau, M.T., Ng, E.H.Y., O, W.S. and Ho, P.C. (2001) Color Doppler analysis of peri-implantation utero-ovarian haemodynamics in women having excessively high oestradiol concentrations after ovarian stimulation. Hum. Reprod., 16, 2114–2117.[Abstract/Free Full Text]

Bhal, P.S., Pugh, N.D., Chui, D.K., Gregory, L., Walker, S.M. and Shaw, R.W. (1999) The use of transvaginal power Doppler ultrasonography to evaluate the relationship between perifollicular vascularity and outcome in in-vitro fertilization treatment cycles. Hum. Reprod., 14, 939–945.[Abstract/Free Full Text]

Bhal, P.S., Pugh, N.D., Gregory, L., O’Brien, S. and Shaw, R.W. (2001) Perifollicular vascularity as a potential variable affecting outcome in stimulated intrauterine insemination treatment cycles: a study using transvaginal power Doppler. Hum. Reprod., 16, 1682–1689.[Abstract/Free Full Text]

Borini, A., Maccolini, A., Tallarini, A., Bonu, M.A., Sciajno, R. and Flamigni, C. (2001) Perifollicular vascularity and its relationship with oocyte maturity and IVF outcome. Ann. NY Acad. Sci., 943, 64–67.[Abstract/Free Full Text]

Chui, D.K.C., Pugh, N.D. and Walker, S.M. (1997) Follicular vascularity—the predictive value of transvaginal power Doppler ultrasonography in an in-vitro fertilization programme: a preliminary study. Hum. Reprod., 12, 191–196.[ISI][Medline]

Dickey, R.P. (1997) Doppler ultrasound investigation of uterine and ovarian blood flow in infertility and early pregnancy. Hum. Reprod. Update, 3, 467–503.[Abstract/Free Full Text]

Gelety, T.J. and Buyalos, R.P. (1993) The effect of clomiphene citrate and menopausal gonadotrophins on cervical mucus in ovulatory cycles. Fertil. Steril., 60, 471–476.[ISI][Medline]

Huey, S., Abuhamad, A., Barroso, G., Hsu, M.I., Kolm, P., Mayer, J. and Oehninger, S. (1999) Perifollicular blood flow Doppler indices, but not follicular pO2, or pH, predict oocyte developmental competence in in-vitro fertilization. Fertil. Steril., 72, 707–712.[ISI][Medline]

Hughes, E., Collins, J. and Vandekerckhove, P. (2000) Clomiphene citrate for unexplained subfertility in women. Cochrane Database Syst. Rev., 2000, 2, CD000057.

Hull, M.G.R., Glazener, C.M.A., Kelly, N.J., Conway, D.I., Foster, P.A., Hinton, R.A., Coulson, C., Lambert, P.A., Watt, E.M. and Desai, K.M. (1985) Population study of causes, treatment, and outcome of infertility. Br. Med. J., 291, 1693–1697.[ISI][Medline]

Marinko, M.B., Neal, G.M., Togas, T. and Tan, S.L. (1999) Prospective randomized double-blind trial of the correlation between time of administration and antiestrogenic effects of clomiphene citrate on reproductive end organs. Fertil. Steril., 71, 633–638.[ISI][Medline]

Massai, M.R., de Ziegler, D., Lesobre, V., Bergeron, C., Frydman, R. and Bouchard, P. (1993) Clomiphene citrate affects cervical mucus and endometrial morphology independently of the changes in plasma hormonal levels induced by multiple follicular recruitment. Fertil. Steril., 59, 1179–1186.[ISI][Medline]

Randall, J.M. and Templeton, A.A. (1991) Transvaginal sonographic assessment of follicular and endometrial growth in spontaneous and clomiphene citrate cycles. Fertil. Steril., 56, 208–212.[ISI][Medline]

Royal College of Obstetricians and Gynaecologists (1998) Evidence-based Clinical Guidelines, No. 2, The Initial Investigation and Management of the Infertile Couple. RCOG Press, London, pp. 141–143.

Tohma, H., Hasegawa, I., Sekizuka, N. and Tanaka, K. (1997) Uterine blood flow. Assessment in an intrauterine insemination program for unexplained infertility. J. Reprod. Med., 42, 463–466.[ISI][Medline]

Zaidi, J., Campbell, S., Pittrof, R. and Tan, S.L. (1995) Endometrial thickness, morphology, vascular penetration and velocimetry in predicting implantation in an in vitro fertilization program. Ultrasound Obstet. Gynecol., 6, 191–198.[ISI][Medline]

Submitted on May 5, 2002; accepted on July 24, 2002.





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