Utero-ovarian blood flow characteristics of pituitary desensitization

Tunde Dada1,3, Osama Salha1, Vicki Allgar2 and Vinay Sharma1

1 Assisted Conception Unit and 2 Department of Research and Development, St James's University Hospital, Leeds, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Down-regulation in assisted reproduction treatment cycles is monitored by suppression of ovarian/pituitary hormones and/or measurement of endometrial thickness. METHODS: This prospective longitudinal study reports on utero-ovarian characteristics of pituitary desensitization. A total of 75 patients were recruited; 32 had IVF treatment, 20 frozen–thawed embryo transfer cycles and 23 patients were recipients of donated oocytes. All received early follicular-phase down-regulation and had colour flow Doppler velocimetry of the utero-ovarian arteries <=3 days before the start of menses and after 21 days of gonadotrophin-releasing hormone (GnRH) analogue treatment. Ovarian volume, endometrial thickness, pituitary and ovarian hormone concentrations were recorded at each scan. RESULTS: Significant changes (P < 0.05) were noted in these and utero-ovarian vasculature during the down-regulation period, with good correlation between resistance index and oestradiol estimations. Neither the type of GnRH analogue nor age influenced the changes in utero-ovarian blood flow. Ovarian artery resistance index was the best Doppler predictor for pituitary suppression and a mean discriminatory cut-off value of 0.867 ± 0.025 was found to have the highest specificity and positive predictive value. CONCLUSIONS: This study has, for the first time, defined cut-off values for satisfactory pituitary suppression with high positive predictive value and specificity in an early follicular phase long protocol of GnRH analogue down-regulation using colour flow Doppler.

Key words: assisted reproduction/Doppler ultrasound/pituitary desensitization/utero-ovarian blood flow


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pituitary desensitization with gonadotrophin-releasing hormone (GnRH) analogues prior to ovarian stimulation, enhances pregnancy rates after IVF and embryo transfer (Serafini et al.1988Go; Hughes et al.1992Go) and is widely employed. Currently successful pituitary down-regulation is assessed by suppression of pituitary and ovarian hormones and/or measurement of endometrial thickness.

More recently however, the use of transvaginal sonography and pulsed colour Doppler velocimetry has made it possible to identify and quantify blood flow in pelvic vessels with satisfactory reproducibility (Tekay and Joupilla, 1996Go). Colour flow Doppler (CFD) studies have demonstrated a correlation between pelvic blood flow and serum oestrogen and progesterone (Jauniaux et al.1992Go; Glock and Brumsted, 1995Go). They have also been used to investigate the relationship between pelvic blood flow and IVF outcome (Weiner et al.1993Go; Tekay et al.1995Go; Bloechle et al.1997Go; Engmann et al.1999Go). Hormonal changes that occur during down-regulation may modulate ovarian and uterine blood flow through their action on oestrogen receptors present on smooth muscle cells on pelvic vasculature (Berquist et al., 1993) and can also be analysed with Doppler velocimetry.

Furthermore as cycle monitoring in assisted reproduction evolves there is a need for less invasive more ultrasound-based programmes, which will reduce overall costs from biochemical testing. In the latter stages of cycles, the role of CFD has already been investigated in monitoring folliculogenesis and oocyte recovery (Balakier and Stronell, 1994Go; Oyesanya et al.1996Go; Nargund et al.1996Go). Studies exclusively on the effects of GnRH analogues on pelvic blood flow in assisted reproduction treatment cycles are lacking.

In this prospective longitudinal study, the blood flow changes that occur at down-regulation after GnRH analogue usage were investigated to establish whether colour flow Doppler can be used to monitor satisfactory pituitary suppression.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This prospective longitudinal study was carried out on 75 consecutive patients who were undergoing assisted reproduction treatment between January 1998 and October 1998 in the Assisted Conception Unit at St James's University Hospital, Leeds, UK, and was approved by our institution's local ethics committee. The mean age of the patients was 33.2 years (range 28–42 years). Thirty-two patients had IVF treatment, 20 patients frozen–thawed embryo transfer cycles and 23 patients were recipients for oocyte donation cycles. No patients with polycystic ovaries were included in the study because polycystic ovaries are associated with different ovarian and uterine haemodynamics when compared with non-polycystic ovaries (Zaidi et al.1998Go). All patients had a body mass index (BMI) of <=32kg/m2, with no systemic or pelvic disease, nor were any patients with ovarian cysts >10 mm included at the baseline scan. All recipients of donated oocytes had not received any hormonal therapy 2 months prior to their treatment cycle. The patients in the study underwent the same long protocol of down-regulation. This involved pituitary desensitization with either buserelin acetate (Hoechst, Hounslow, Middlesex, UK) (n = 23), at a dose of 0.5 mg s.c. daily or nafarelin intranasal spray (Searle, High Wycombe, Bucks, UK), (n = 52), 200 µg 8 hourly from the first day of the menstrual cycle and continued for 21 days. Use of either analogue was decided by patient preference.

The baseline scan was performed on each patient within 3 days of the start of the ongoing treatment menstrual cycle and the pre-stimulation scan after 21 days of down-regulation with a GnRH analogue. The following were recorded: (i) the ovarian volume (both right and left ovaries), (ii) endometrial thickness and (iii) Doppler estimations of the (right and left) ovarian arteries and the mean (right and left) ascending uterine arteries as described below.

A venous blood sample was also obtained for estimation of serum oestradiol, progesterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at the time of scanning.

There is at present no definitive data for optimum oestradiol concentrations after the down-regulation phase and cut-off values vary widely (Porcu et al.1995Go; Bloechle et al.1997Go; Barash et al.1998Go). Satisfactory down-regulation was defined by a cut-off value for oestradiol of <=130 pmol/l (~35 pg/ml) (Dada et al.1999Go).

Transvaginal colour Doppler velocimetry
Interobserver variability was eliminated by a single operator (TD) scanning all the patients in this study and all Doppler scans were carried out at the same time of day (0800–1200 h) to eliminate diurnal variation (Zaidi et al.1995Go).

All scans were performed in the lithotomy position with an empty bladder utilizing a 5 MHz transvaginal probe with colour and pulsed Doppler facilities (Aloka SSD-1700; Aloka Corporation, Tokyo, Japan). A pilot study of 20 patients was performed to determine reproducibility of blood flow measurements in the ovarian and uterine vessels. In this study each patient was scanned as described above and uterine and ovarian artery Doppler measurements obtained. The operator was blinded to these results and the patients rescanned after ~5 min (Tekay and Joupilla, 1996Go). The coefficient of variation (CV) for the ovarian and uterine flows was <12% for all Doppler parameters and the interclass correlation coefficients (ICC) ranged between 0.86–0.98 for both arteries (Bland and Altman, 1992Go).

For all Doppler scans, the high pass wall filter was set at 50 Hz to eliminate low frequency signals and the pulse repetition frequency at 2–10 kHz. The spatial peak temporal average intensity (SPTA) was <80 mW/cm2, within safety limits set by the American Institute of Ultrasound in Medicine (AIUM, 1992).

Initially B-mode grey scale sonography was used to examine ovarian and uterine morphology. The ovarian volume was calculated from three maximum measurements of: the longitudinal (D1), antero-posterior (D2) and transverse (D3) diameters, scanning in sagittal and coronal planes. The ovarian volume was obtained using the formula for the volume of a prolate ellipsoid: D1xD2xD3x0.52. Endometrial thickness was calculated as the distance between the superior and inferior endometrial–myometrial interface, excluding the echoless zone.

The ovarian arteries were identified at the superio-lateral border of each ovary with colour flow and the signal interrogated with pulsed Doppler (Figure 1Go).Occasionally the ovarian artery signal was not clear and the sample volume was moved across the ovary until a clear signal was identified. The ovarian signals were recognized by low velocity and high impedance helping to distinguish them from the other vessels in proximity (Kurjak and Kupesic, 1995Go). Each ascending branch of the uterine artery was identified, lateral to the cervix and waveform patterns generated. In all Doppler scans alteration of the angle of insonation produced maximum colour density so that at least three waveform patterns were analysed for measurement of the pulsatility index (PI), resistance index (RI), peak systolic velocity (PSV) and the time averaged maximum velocity (TAMX), which were all determined by the ultrasound software.



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Figure 1. Identification of the ovarian artery by pulsed Doppler.

 
Hormone assays
Serum FSH and LH were measured in all samples using a Technicon Immuno 1 automated clinical analyser employing a sandwich immunoassay format, with a detection limit of 0.1 mIU/l (Bayer Diagnostics, Newbury, Berks, UK). Serum oestradiol and progesterone assays used a similar kit (Bayer Diagnostics) employing a competitive immunoassay format with a detection limit of 37 pmol/l and 0.4 nmol/l respectively. The inter-assay CV for FSH, LH, oestradiol and progesterone were 4, 4.6, 6.2 and 7.8% while the intra-assay CV were 3.2, 4.5, 5.1 and 9.3% respectively.

Statistical analysis
Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS) version seven and results expressed as mean ± SD. Analysis of variance (ANOVA) of replicate measurements was employed for reproducibility data. The Student's t-test was used to test for equality of means, the Wilcoxon matched-pairs signed rank test for non-normally distributed data and ANOVA to compare age groups. Pearson's correlation was used to investigate the relationship between Doppler impedance and oestradiol, linear regression to study predictability of Doppler measurements from oestradiol concentrations and multiple regression to stratify the most important factors in down-regulation. To predict best suppression cut-off values, receiver operating characteristic (ROC) analysis was used and sensitivities and specificities calculated. A P value of <0.05 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Doppler waveforms for evaluation were obtained from all the patients in the study.

The mean ages for the IVF, frozen–thawed embryo transfer and oocyte donation cycles groups of patients were 30.4 ± 4, 31.2 ± 3 and 38.2 ± 3 years respectively. Patients undergoing oocyte donation treatment were significantly older (P < 0.05) than the two other groups.

Initial evaluation of right and left ascending uterine arteries showed no significant differences between the two and so for the purposes of the study the mean uterine artery flow has been used.

There were no patients with ovarian cysts >10 mm at the baseline scan included in the study and a non-significant increased incidence of remnant corpus luteum (CL) cysts was found in the left ovary compared with the right (53 versus 47%). Also, comparison of blood flow in ovaries with and without remnant CL cysts revealed a marginally higher flow in the ovarian artery on the side of the CL, but this did not reach statistical significance. After down-regulation, there was no significant difference in ovarian artery blood flow between the two groups with or without cysts nor when the percentage change in Doppler characteristics from BL to PS was investigated.

Results of hormone profile, endometrial thickness and ovarian volume changes after down-regulation
Table IGo shows the changes in hormone profile, ovarian volume and endometrial thickness during down-regulation. The mean serum hormone concentrations decreased from baseline to pre-stimulation and these changes were significant in oestradiol, progesterone and LH. Although there was a fall in the mean FSH concentration, this did not reach statistical significance. Mean pre-stimulation oestradiol values were below the defined cut-off value of 130 pmol/l.


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Table I. Comparison of serum hormone concentrations and ovarian volumes at baseline and pre-stimulation
 
The baseline ovarian volumes in both right and left ovaries were comparable as were the post GnRH analogue volumes. There was a significant decrease in ovarian volumes after pituitary desensitization (P < 0.001) in both ovaries and endometrial thickness fell significantly (P < 0.0001) over the down-regulation period.

Comparison of Doppler measurements at baseline and pre-stimulation
Table IIGo shows how Doppler measurements changed over the down-regulation period. Impedance studies (RI and PI) showed a significant (P < 0.05) increase in resistance over the study period while PSV and TAMX revealed an overall significant reduction (P < 0.05) in blood flow velocity. Doppler parameters were similar in the right and left ovarian arteries with no significant difference between the two.


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Table II. Comparison of baseline and pre-stimulation Doppler characteristics in the ovarian and uterine arteries
 
Pearson's correlation coefficients showed a significant association between RI and serum oestradiol at BL (r = -0.76; P < 0.05) and PS (r = -0.63; P < 0.05).

Comparison of GnRH analogues
The percentage changes in ovarian and uterine blood flow were compared after 21 days of down-regulation with nafarelin (n = 23) versus buserelin (n = 52) administration. There were no significant differences in the changes in blood flow between buserelin and nafarelin when all Doppler parameters were compared. Figure 2Go shows the mean percentage change in Doppler measurements in ovarian vasculature.



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Figure 2. Comparison of the mean percentage change in Doppler measurements from baseline to pre-stimulation with nafarelin (NAF) versus buserelin (BUS) in the ovarian vasculature. There were no significant differences between the two analogues. RI = resistance index; PI = pulsatility index; PSV = peak systolic velocity; TAMX = time average maximum velocity.

 
Comparison of age groups
Table IIIGo shows the effect of age on the Doppler measurements in the ovarian and uterine arteries. There was no significant difference in either the baseline or pre-stimulation Doppler PI or PSV when comparing the age groups of <=30, 31–35 or >35 years.


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Table III. Comparison of pulsatility index and peak systolic velocity in different age groups
 
Developing discriminatory cut-off values for satisfactory down-regulation
Tables IVa and bGoGo, list the results of receiver operating characteristic (ROC) curves calculated for each of the parameters:– endometrial thickness (ET), the hormones (FSH, LH, progesterone), ovarian volume (OV right and left) and the Doppler parameters (RI, PI, PSV and TAMX), for the right and left ovarian and uterine arteries. Each table demonstrates the relationship between sensitivity and specificity for satisfactory down-regulation using oestrogen concentrations of <=130 pmol/l. At this oestradiol value the best cut-off desensitization values were calculated for the tests above.


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Table IVa. Desensitization cut-off values for endometrial thickness, ovarian and pituitary hormones and ovarian volume
 

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Table IVb. Desensitization cut-off values for Doppler parameters in the right and left ovarian arteries and the uterine artery
 
The results show that in this study population, ET and Doppler resistance indices appear to be the best tests, with RI superior to PI.

An ideal test with 100% sensitivity and 100% specificity would have an area under the ROC curve of 1. If the test has no discriminatory power whatever, the ROC curve will be a straight line and the area under the curve will be 0.5 and at any given cut-off value the chances of a true-positive and a false-positive would be equal. With a sensitivity of ~90% and positive predictive value of ~97%, the resistance index discriminatory cut-off values for satisfactory suppression of ovarian blood flow in the right and left ovarian arteries was 0.841 and 0.805 respectively (mean 0.823 ± 0.018).

However regarding ovarian desensitization in IVF cycles, it is imperative that patients not adequately suppressed are identified. This would prevent continuation of the treatment cycle before satisfactory down-regulation occurs. It is therefore more important to have high specificity than sensitivity in developing cut-off values for test values. Increasing specificity to 100% (i.e. identifying all non-adequately suppressed women with a low false positive rate) decreases sensitivity of the tests, with the result of altered cut-off values of 0.892 and 0.842 (mean 0.867 ± 0.025) as shown in Table VGo.


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Table V. Best discriminatory cut-off values for Doppler measurements with 100% specificity in the right and left ovarian arteries and uterine arteries
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pituitary desensitization has become an integral part of assisted reproduction treatment, yet not enough is known about the changes in the blood flow of pelvic organs that are associated with this important phase of the cycle.

This study demonstrates that all Doppler parameters measured in the ovarian and uterine vessels are significantly altered with GnRH analogues and that when the RI, PI, PSV and TAMX are compared, RI has the highest sensitivity and specificity for satisfactory down-regulation in the ovarian vasculature.

Ovarian blood flow can be studied either as the ovarian artery courses towards the substance of the ovary (Kurjak and Kupesic, 1995Go) or by analysing the artery within the ovary itself, i.e. intra-ovarian/stromal flow. The technique for measuring intra-ovarian blood flow is well described and involves assessing each ovary by studying `any small artery in the ovarian stroma not close to the surface of the ovary or near the wall of a follicle' (Zaidi et al.1996Go). No correction is made for the angle of insonation, because it cannot be determined for such small vessels. Hence the reproducibility of pulsatility index measurements in intra-ovarian vessels has been questioned (Tekay and Joupilla, 1996Go).

The reports of GnRH analogue effects on intra-ovarian blood flow has not been consistent. Tekay and colleagues were able to detect intra-ovarian blood flow after 2 weeks of pituitary suppression with buserelin acetate in 10 out of 29 cases (34%) (Tekay et al.1995Go), whereas Lunenfeld and co-workers (1996) detected intra-ovarian flow in only 9% of patients down-regulated with a GnRH analogue (Lunenfeld et al., 1996Go). For the above reasons only ovarian artery blood flow was investigated in the current study. It has been possible to obtain satisfactory reproducibility by eliminating inter-observer and diurnal variation and eliminating any side bias by studying the right and left ovarian arteries independently.

By contrast, the measurement of uterine artery blood flow in most studies is generally uniform. The vessels are large and readily accessible allowing for ease of identification. Proof that GnRH agonists affect blood flow has already been established (Matta et al.1988Go; Battaglia et al.1995Go). GnRH analogue administration to women with uterine fibroids has been shown to significantly increase uterine artery RI after 2–4 months of therapy (Aleem and Predanic, 1995Go; Shaw et al.1996).

Agonist treatment creates a state of gonadotrophin desensitization and ovarian suppression with a subsequent hypo-oestrogenic state. Receptors for GnRH have been identified both in the ovary and the endometrium (Broekmans, 1996Go), but not in ovarian and uterine artery endothelium. Conversely, oestrogen receptors are widely distributed on the smooth muscle cells of vessels (Perrot-Applanat, 1988). Studies comparing pelvic blood flow with serum oestrogen concentrations have been equivocal. Some have shown a good correlation (Goswamy et al.1988; de Ziegler et al., 1991; Weiner et al.1993Go; Lunenfeld et al.1996Go) while others found a poor correlation (Tekay et al.1995Go). In the current study linear regression analysis showed a significant correlation (P < 0.05) between serum oestradiol and RI values in the ovarian arteries after down-regulation. Nevertheless multiple regression analysis of all the factors investigated showed endometrial thickness to be the most accurate independent predictor of pituitary suppression as measured by hypo- oestrogenism (oestradiol <130 pmol/l). RI of the ovarian arteries was the next best predictor and although the multiple correlation coefficient statistic increased with the additive effect of RI and endometrial thickness, this was not statistically significant. Although some direct effect for GnRH analogues can thus be postulated by the changes in utero-ovarian blood flow, it can be safely assumed to be principally a result of the hypo-oestrogenism caused by pituitary desensitization. Furthermore though there may have been possible bias in evaluating baseline parameters <=3 days from the start of GnRH analogue administration we do not believe this to be significant because of the short time interval.

This study confirms that the overall effect of GnRH analogues is to reduce blood flow to the target organs, mainly the uterus and ovaries (Table IIGo). This finding contradicts a report (Cacciatore et al., 1990Go) where no change in uterine blood flow after 2–4 weeks of down-regulation in their study was detected. Paradoxically another study (Bassil et al., 1995Go) describes low uterine RI values after 2–3 weeks of down-regulation with an increase after the fourth week, despite the commencement of gonadotrophin stimulation.

In later work (Bassil et al.1997Go) the authors were able to demonstrate a low mean intra-ovarian RI of 0.53 after the same period of pituitary suppression, with an increase in ovarian artery resistance after initiating human menopausal gonadotrophin (HMG). They concluded that administration of GnRH analogue increases the ovarian RI. However the increase in RI occurred during gonadotrophin administration and increased oestrogen secretion, somewhat contrary to the known and established effects of oestrogen on smooth vessel musculature and blood flow. Oestrogens have been shown to cause vasodilatation and decreased impedance to blood flow (Resnik et al.1974Go; Hutchison et al.1997Go; Cagnacci et al.2000Go). Furthermore, in the study by Bassil et al. (1997) no values for Doppler measurements were recorded prior to analogue administration and thus it is difficult to determine how the increase in RI as a direct result of GnRH analogue was calculated.

The results of the current study agree in part with other work (Engmann et al.1999Go). This group was able to show a significant decline in ovarian stromal flow velocity after 2–3 weeks of GnRH agonist administration but were unable to show a similar reduction in the uterine blood flow during the same period. The results presented here are more in agreement with Battaglia et al. (1997), who demonstrated an increase in uterine artery PI after down-regulation with GnRH analogue in an early follicular phase long protocol of analogue administration (Battaglia et al., 1997Go). A significantly increased ovarian vascular impedance after 14 days of GnRH analogue administration in women without polycystic ovaries (PCO) was demonstrated (Vrtacnik-Bokal and Meden-Vrtovec, 1998Go).

As stated above, the current study demonstrated that RI in the ovarian artery is probably the most accurate Doppler parameter in assessing satisfactory down-regulation. The mean ovarian artery RI cut-off value of 0.867 ± 0.025 would seem to be of greater significance in clinical practice when all unsatisfactorily down-regulated patients need to be identified. Uterine artery discriminatory tests had uniformly poorer sensitivity at the same concentration of specificity than for the ovarian arteries and this may be due to the fact that these larger arteries may be less sensitive to the hormonal effects of GnRH analogues.

The changes in ovarian volume that occur over the down-regulation period were also investigated. Ovarian volume as a measure of ovarian reserve has been shown to be predictive of IVF outcome (Syrop et al.1995Go; Lass et al.1997Go). A significant decrease in ovarian volume in both the right and left ovaries after GnRH analogue administration (P < 0.001) was shown. This finding is not in agreement (Sharara et al.1999Go), where ovarian volume changes were studied in 38 patients pre-treated with oral contraceptives for 3 weeks (range 3–6 weeks), prior to leuprolide acetate (LA) administration for 21 days. They concluded that pituitary desensitization with LA had no effect on ovarian volume measurements, but their cohort of patients already had suppressed inactive ovaries due to the oral contraceptive pill and they had not excluded women with PCO. In two studies (Farquhar et al.1994Go; Wu et al.1998Go), it has been shown that patients with PCO have larger ovaries than non-PCO controls. Furthermore it has been shown that oral contraception significantly reduces ovarian volume (Christensen et al.1997Go). These confounding factors may have introduced bias into their ovarian volume measurements.

Studies investigating predictors of IVF treatment outcome have shown a relationship between increased female age, higher basal FSH concentrations and reduced ovarian volume with an overall poorer outcome (Toner et al., 1991Go; Scott and Hoffman, 1995Go). In the current study no significant correlation was found between the change in ovarian volume with down-regulation and the change in Doppler measurements over the same time period. Whether percentage reduction in ovarian volume and Doppler measurements with GnRH analogues can be used prognostically in assessing ovarian reserve remains to be investigated.

In a previous report it was shown that by 21 days there is comparable suppression of ovarian hormones by nafarelin and buserelin (Dada et al.1999Go). In this study it was found that both of these drugs cause similar blood flow changes in both the uterine and ovarian vasculature during the down-regulation phase. Larger study groups are required to confirm these findings.

Female partner's age is an established prognostic indicator of treatment outcome in assisted conception (Check et al.1994Go). In the current study no evidence was found of a more profound analogue suppression (significantly higher PI or depressed PSV values) in the older patients in the study group (Table IIIGo).

In conclusion, all ultrasound parameters studied (including endometrial thickness, ovarian volume and utero-ovarian blood flow), changed significantly after pituitary down-regulation. Endometrial thickness accurately and independently predicted desensitization with ovarian artery RI the next best predictor. In this study, for the first time, cut-off values have been defined for ovarian artery blood flow, with a high positive predictive value and specificity for satisfactory pituitary suppression in an early follicular phase long protocol of GnRH analogue down-regulation using colour flow Doppler.


    Notes
 
3 To whom correspondence should be addressed at: Assisted Conception Unit, St James's University Hospital, Leeds LS9 7TF, UK. E-mail: Babs{at}tdada.freeserve.co.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 13, 2000; accepted on March 30, 2001.