Endometrial and subendometrial blood flow measured during early luteal phase by three-dimensional power Doppler ultrasound in excessive ovarian responders

Ernest Hung Yu Ng1,2, Carina Chi Wai Chan1, Oi Shan Tang1, William Shu Biu Yeung1 and Pak Chung Ho1

1 Department of Obstetrics and Gynaecology, The University of Hong Kong, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong, People’s Republic of China

2 To whom correspondence should be addressed. e-mail: nghye{at}hkucc.hku.hk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Impaired implantation in assisted reproduction cycles with high serum estradiol (E2) concentrations may be related to suboptimal endometrial perfusion. Endometrial and subendometrial blood flow were compared between excessive responders (serum E2 on the day of HCG >20 000 pmol/l) and moderate responders (E2 ≤20 000 pmol/l). METHODS: Three-dimensional (3D) ultrasound examination with power Doppler was performed 2, 4 and 7 days after HCG in 32 patients who did not have embryo transfer in order to measure endometrial thickness, pulsatility index (PI)/resistance index (RI) of uterine vessels, and endometrial volume, vascularization index (VI)/flow index (FI)/vascularization flow index (VFI) of endometrial and subendometrial regions. RESULTS: Excessive responders tended to have lower endometrial and subendometrial VI/VFI on HCG +2 and more absent endometrial/subendometrial blood flow. They had significantly higher endometrial FI and subendometrial VFI than moderate responders on HCG +7. Only in the excessive responder group, uterine PI/RI declined significantly from HCG +2 to HCG +7 and endometrial VI/VFI increased significantly from HCG +4 to HCG +7. CONCLUSION: Changes in uterine Doppler flow indices, and endometrial and subendometrial 3D power Doppler flow indices during the early luteal phase were significantly different between moderate and excessive responders.

Key words: endometrial and subendometrial blood flow/excessive responders/three-dimensional power Doppler


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
High serum estradiol (E2) concentrations resulting from excessive ovarian responses adversely affect the outcomes of assisted reproduction cycles (Forman et al., 1988Go; Simón et al., 1995Go, 1998; Check et al., 2000Go; Ng et al., 2000aGo). Although such adverse effects were not observed in some studies (Chenette et al., 1990Go; Gelety and Buyalos, 1995Go; Sharara and McClarmrock, 1999Go; Papageorgiou et al., 2002Go), we have clearly shown significant impairment in pregnancy rates in stimulated cycles when the serum E2 concentration on the day of HCG was >20 000 pmol/l (Ng et al., 2000aGo). Oocyte and embryo quality were not affected by the high serum E2 concentrations (Ng et al., 2003Go).

Therefore, we postulate that reduced implantation in those cycles with excessively high serum E2 concentrations is most probably related to an adverse environment in the endometrium. A rapid decline in serum E2 around the peri-implantation period (Ng et al., 2000bGo) could not explain the impaired uterine receptivity, which may be partially attributed to abnormal endometrial morphometry in the form of delayed glandular maturation and advanced stromal morphology (Basir et al., 2001aGo). Angiogenesis plays a critical role in various female reproductive processes such as development of a dominant follicle, formation of a corpus lutuem, growth of endometrium and implantation (Abulafia and Sherer, 2000Go; Smith, 2001Go). A good blood supply is usually considered to be an essential requirement for normal implantation. Gannon et al. (1997Go) used an intrauterine laser Doppler technique to measure endometrial microvascular blood flow, which varied throughout the menstrual cycle, with increases in blood flow during early follicular and early luteal phase. Endometrial blood flow measured by this technique in the early luteal phase of the cycle preceding the IVF cycle has been shown to be predictive of pregnancy and superior to other conventional parameters predicting endometrial receptivity (Jinno et al., 2001Go). Suboptimal endometrial blood flow in excessive responders may be another reason for reduced endometrial receptivity.

We previously assessed uterine and endometrial perfusion of excessive responders by colour Doppler ultrasound on the day of embryo transfer (Basir et al., 2001bGo). Despite significantly lower uterine Doppler flow indices, the number of patients from the excessive responder group showing endometrial colour signals from the pulsatile vessels in the endometrium was significantly lower than the number in the moderate responder group, i.e. those with serum E2 ≤20 000 pmol/l on the day of HCG. More recently, blood flow in the endometrium and the subendometrial regions can be determined by the use of three-dimensional (3D) ultrasound with power Doppler (Schild et al., 2000Go; Kupesic et al., 2001Go; Wu et al., 2003Go). Power Doppler imaging is more sensitive than colour Doppler imaging at detecting low velocity flow, and hence improves the visualization of small vessels (Guerriero et al., 1999Go). The aim of this study was to compare endometrial and subendometrial blood flow measured by two-dimensional transvaginal and 3D power Doppler ultrasound during the early luteal phase between patients with serum E2 concentration on the day of HCG of >20 000 and ≤20000 pmol/l.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infertile patients attending the Assisted Reproduction Unit at the Department of Obstetrics and Gynaecology, University of Hong Kong, for IVF/embryo transfer (ET) treatment were recruited when ET was not performed as a result of absence of spermatozoa in testicular sperm extraction, failure of fertilization due to male factors or serum E2 levels on the day of HCG >20 000 pmol/l. All fresh embryos were cryopreserved in patients with serum E2 levels on the day of HCG >20 000 pmol/l because of possible risks of ovarian hyperstimulation syndrome (OHSS) and a reduced pregnancy rate in these stimulated cycles. Every patient gave written informed consent prior to participating in the study, which was approved by the Ethics Committee, Faculty of Medicine, University of Hong Kong. Each patient was evaluated only once during the study period and did not receive any monetary compensation for participation in this study.

All patients received a long protocol of pituitary downregulation as previously described (Ng et al., 2000aGo). In brief, all women were pre-treated with buserelin (Suprecur, Hoechst, Frankfurt, Germany) nasal spray 150 µg four times a day from the midluteal phase of the cycle preceding the treatment cycle and received HMG (Pergonal, Serono, Geneva, Switzerland) for ovarian stimulation. HCG (Profasi, Serono, Geneva, Switzerland) 10 000 IU was given i.m. when the leading follicle reached 18 mm in diameter and there were at least three follicles of ≥16 mm in diameter. Blood was taken on the day of HCG administration for serum E2 concentration. Serum E2 concentrations were measured using commercially available kits (Automated Chemiluminescence System, Bay Corporation, NY). The intra- and inter-assay coefficients of variation were 8.1 and 8.7%, respectively.

Measurement of uterine, endometrial and subendometrial blood flow
Ultrasound measurement of all patients was performed by E.H.Y.N. on HCG +2 days (prior to transvaginal ultrasound-guided oocyte retrieval), +4 days and +7 days, using Voluson 730® (Kretz, Zipf, Austria) at ~8–10 a.m. after they had emptied their bladder. The endometrial thickness was measured in a longitudinal plan. Using colour Doppler in the two-dimensional mode, flow velocity waveforms were obtained from the ascending main branch of the uterine artery on the right and left side of the cervix before it entered the uterus in a longitudinal plane. 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. The intra-observer coefficient of variation is 5.6% for endometrial thickness, 9.6% for PI and 4.1% for RI.

The ultrasound machine was then switched to the 3D mode with power Doppler. The setting condition for this study was: frequency, mid; dynamic set, 2; balance, G>140; smooth, 5/5; ensemble, 12; line density, 7; power Doppler map, 5; and the setting condition for the subpower Doppler mode was: gain, –6.0; balance, 140; quality, normal; wall motion filter, low 1; velocity range, 0.9 kHz. The resultant truncated sector covering the endometrial cavity in a longitudinal plane of the uterus was adjusted and moved, and the sweep angle was set to 90° to ensure that a complete uterine volume encompassing the entire subendometrium was obtained. The patient and the 3D transvaginal probe remained as still as possible during the volume acquisition. A 3D data set was then acquired using the medium speed sweep mode. The resultant multiplanar display was examined to ensure that the area of interest had been captured in its entirety. If the volume measurement was complete without power Doppler artefact, the data set was stored for later analysis by E.H.Y.N. If there was apparent artefact, such as typical ‘flash’ artefacts seen with bowel movements, the data set was reacquired until a satisfactory image was obtained.

The built-in VOCAL® (virtual organ computer-aided analysis) Imaging Program for the 3D power Doppler histogram analysis was used together with computer algorithms to form the endometrial volume and indices of blood flow within the endometrium. The vascularization index (VI) indicated the proportion of the volume showing a flow signal in the total volume of the endometrium. The flow index (FI) was an average of the intensity of flow signal inside the endometrium. The vascularization flow index (VFI) was a combination of the presence of the vessel and the amount of flow produced by multiplying FI and VI (Pairleitner et al., 1999Go). During the analysis and calculation, the manual mode of the VOCAL® Contour Editor was used to cover the whole 3D volume of the endometrium with a 15° rotation step. Hence, 12 contour planes were analysed for the endometrium of each patient to cover 180°. Following assessment of the endometrium itself, the subendometrium was examined through the application of ‘shell-imaging’, which allows the user to generate a variable contour that parallels the originally defined surface contour. In this study, the subendometrial region was considered to be within 1 mm of the originally defined myometrial–endometrial contour. VI, FI and VFI of the subendometrial region were obtained accordingly.

The intra-observer variations for endometrium volume, VI, FI and VFI were 4.2, 9.1, 1.3 and 9.3%, respectively. The readings of the two independent assessments by the same investigator were well correlated, with the Pearson’s correlation coefficients for endometrial volume, VI, FI and VFI being 0.99, 0.98, 0.99 and 0.97, respectively.

Statistical analysis
Patients with serum E2 ≤20 000 and >20 000 pmol/l were considered as moderate and excessive responders, respectively. The primary outcome measures were VI, FI and VFI of endometrial and subendometrial regions. Continuous variables were not normally distributed and were given as median (range), unless indicated. Statistical comparison was carried out by Mann–Whitney, {chi}2 and Fisher’s exact tests, where appropriate. Repeated measurements were first analysed with the Friedman test, and any difference between paired data was compared by Wilcoxon signed ranks test. A P-value (two-tailed) of <0.05 was taken as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 32 consecutive patients were recruited from June 2002 to September 2003: 15 moderate responders and 17 excessive responders. All patients had no distortion of the endometrial lining confirmed in the multiplanar display and had a triple layer pattern of the endometrium on HCG +2. They did not suffer from a moderate or severe degree of OHSS (RCOG Guideline No 5, 1995Go).

Moderate responders versus excessive responders
Table I summarizes the demographic data and ovarian responses. There were no differences in age of women, primary/secondary infertility, cause of infertility, presence of fibroids, duration of infertility, and HMG dosage and duration between moderate and excessive responders. Excessive responders had a significantly lower body mass index and a lower basal FSH level, but more oocytes aspirated than moderate responders.


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Table I. Comparison of demographic data and ovarian responses between moderate and excessive responders
 
Compared with moderate responders, excessive responders had significantly higher endometrial thickness on HCG +2 (median: 13.7 mm versus 10.0 mm, respectively, P = 0.018, Mann–Whitney test) and significantly lower uterine PI on HCG +7 (median 1.55 versus 1.99, respectively, P = 0.027, Mann–Whitney test). There were only non-significant trends of higher endometrial thickness on HCG +4 and +7, and of lower PI on HCG +2 and +4 in the excessive responder group (Figure 1). Endometrial volume and uterine RI did not differ between excessive and moderate responders on HCG +2, +4 and +7.



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Figure 1. Boxplot of endometrial thickness, endometrial volume, uterine PI and RI during early luteal phase in moderate and excessive responders (open box HCG +2; diagonal hatching HCG +4; vertical hatching HCG +7).

 
Excessive responders had significantly higher endometrial FI (median: 22.17 versus 21.39, respectively, P = 0.042, Mann–Whitney test) and subendometrial VFI (median: 0.77 versus 0.32 respectively, P = 0.029, Mann–Whitney test) on HCG +7, when compared with moderate responders (Figure 2). There were non-significant trends of lower endometrial and subendometrial VI/VFI on HCG +2 but higher endometrial VI/VFI and subendometrial VI on HCG +7 in excessive responders. The number of patients with absent endometrial or subendometrial blood flow during the early luteal phase tended to be higher in excessive responders than in moderate responders, but the difference did not reach statistical significance (Table II).



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Figure 2. Boxplot of endometrial and subendometrial blood flow during early luteal phase in moderate and excessive responders (open box HCG +2; diagonal hatching HCG +4; vertical hatching HCG +7).

 

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Table II. Absent endometrial or subendometrial blood flow during the early luteal phase in moderate and excessive responders
 
Changes in endometrial and subendometrial blood flow during the early luteal phase (Figures 1 and 2)
Moderate responders. No differences in endometrial thickness, endometrial volume, uterine PI/RI, endometrial VI/FI/VFI and subendometrial FI were found between HCG +2, +4 and +7. There were significant reductions in subendometrial VI/VFI from HCG +2 to +7.

Excessive responders. No difference in endometrial thickness, endometrial volume, endometrial and subendometrial FI were found between HCG +2, +4 and +7. Uterine PI and RI declined significantly from HCG +2 to HCG +7. Endometrial VI and VFI were significantly higher on HCG +7 than on HCG +4, whereas subendometrial VI and VFI were significantly lower on HCG +4 than on HCG +2 and +7.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The endometrium undergoes adequate proliferation and differentiation in the follicular phase, which is followed by timely secretory changes during the luteal phase. Therefore, endometrial receptivity can be evaluated by histological evaluation of an endometrial biopsy (Noyes et al., 1950Go), endometrial proteins in uterine flushing (Dalton et al., 1995Go; Li et al., 1998Go) or, more commonly, non-invasive ultrasound examination of the endometrium. Different ultrasound parameters have been used to assess endometrial receptivity, including endometrial thickness, endometrial pattern, endometrial volume, Doppler study of uterine arteries and endometrial blood flow. Endometrial thickness and pattern, used extensively in predicting the IVF outcome, have low positive predictive value and specificity (Turnbull et al., 1995Go; Friedler et al., 1996Go), whereas endometrial volume measured by 3D ultrasound is not predictive of pregnancy (Raga et al., 1999Go; Yaman et al., 2000Go; Schild et al., 2001Go). The endometrial thickness was significantly higher in excessive responders on HCG +2. Weissman et al. (1999Go) showed adverse IVF outcomes when endometrial thickness was >14 mm, but such detrimental effect could not be confirmed by others (Yakin et al., 2000Go; Dietterich et al., 2002Go). Endometrial thickness and endometrial volume did not change during the early luteal phase in either excessive or moderate responders.

Blood flow in the uterine blood vessels assessed by colour Doppler ultrasound is usually expressed as downstream impedance to flow because measurement of blood flow volume is difficult and inaccurate, depending on the angle of insonation, accurate measurement of the diameter of vessels and tortuosity of the vessels (Dickey, 1997Go). Steer et al. (1992Go) showed that the chances of pregnancies were maximal when uterine PI was in the range of 2.00–2.99. There was a significant correlation of uterine PI and biochemical markers of endometrial receptivity including endometrial histological dating, and expression of E2 receptor and a 24 kDa protein (Steer et al., 1995Go).

We previously found significantly lower uterine and ovarian PI and RI on HCG +4 (day of ET) in excessive responders than in moderate responders (Basir et al., 2001bGo). In the present study, uterine PI was significantly lower in excessive responders on HCG +7 only, and uterine RI was not different between excessive and moderate responders during the early luteal phase. On the other hand, uterine PI and RI declined significantly during the early luteal phase in excessive responders only. There is still controversy in the literature regarding changes in uterine Doppler flow indices during the luteal phase of a spontaneous cycle. Some studies (Goswamy and Steptoe, 1988Go; Kupesic and Kurjak, 1993Go; Sladkevicius et al., 1993Go; Tan et al., 1996Go) reported that resistance to flow declined from the early follicular phase to the midluteal phase but became maximal around the time of ovulation. Others (Thompson et al., 1988Go; Long et al., 1989Go; Scholtes et al., 1989Go) noted no differences in uterine Doppler flow indices at different phases of the cycle. Periodic variations in uterine PI were not abolished in patients following stimulation with clomiphene citrate or HMG (Kupesic and Kurjak, 1993Go). The continuous decline in uterine PI and RI of excessive responders in the early luteal phase may be related to the effects of sustained high serum E2 levels in these patients, but the pathophysiological significance is not known.

Doppler study of uterine arteries may not reflect the actual blood flow to the endometrium as the major compartment of the uterus is the myometrium and there is collateral circulation between uterine and ovarian vessels. Therefore, it is more logical to determine the endometrial blood flow. Blood flow to the endometrium comes from the radial artery, which divides after passing through the myometrial–endometrial junction to form the basal arteries that supply the basal portion of the endometrium, and the spiral arteries that continue up towards the endometrium. Kupesic and Kurjak (1993Go) first examined spiral vessels by transvaginal colour Doppler ultrasound during the peri-ovulatory period in women undergoing insemination. Contart et al. (2000Go) subjectively graded endometrial flow by the visualization of power Doppler in the quadrants in the fundal region of the transverse plane and Yang et al. (1999Go) measured the area and intensity of colour signals present in the endometrium in a longitudinal axis by computer software. More recently, blood flow in the endometrium and the subendometrial region can be measured objectively by the use of 3D ultrasound with power Doppler (Schild et al., 2000Go; Kupesic et al., 2001Go; Wu et al., 2003Go). Higher endometrial FI on the day of ET was shown in pregnant cycles (Kupesic et al., 2001Go). Subendometrial VFI on the day of HCG was superior to endometrial volume, and subendometrial VI and FI in predicting the IVF outcome (Wu et al., 2003Go).

To the best of our knowledge, this is the first study addressing endometrial and subendometrial blood flow during the early luteal phase measured by 3D power Doppler ultrasound in patients with moderate and excessive ovarian responses during IVF/ET treatment. Endometrial VI and VFI tended to be lower on HCG +2 but higher on HCG +7 in excessive responders than in moderate responders. A similar trend in subendometrial VI and VFI on HCG + 2 and +7 was also seen in excessive responders. A significant difference in subendometrial VFI was demonstrated between excessive and moderate responders on HCG +7 only, probably because of the large variations in these parameters and the small number of subjects in this study. More excessive responders appeared to have absent endometrial or subendometrial blood flow during the early luteal phase than moderate responders, especially on HCG +4. The small number of subjects with absent flow precluded meaningful statistical analysis. Absent endometrial or subendometrial flow detected by colour or power Doppler in two-dimensional ultrasound is associated with no pregnancy (Zaidi et al., 1995Go; Battaglia et al., 1997Go) or a much reduced pregnancy rate (Chien et al., 2002Go; Maugey-Laulon et al., 2002Go).

The longitudinal observations in the early luteal phase revealed interesting findings. Endometrial and subendometrial VI/VFI was significantly increased in excessive responders by HCG +7 and the increase in endometrial and subendometrial blood flow may be related in part to the continuous decline in uterine PI and RI in this group. On the other hand, endometrial VI/VFI did not change and subendometrial VI/VFI declined gradually in moderate responders during the early luteal phase. Morphometric analysis of endometrial biopsies taken on HCG +7 revealed that the number of blood vessels per mm2 was significantly higher in excessive responders than in moderate responders (Basir et al., 2001aGo). This morphometric difference may offer another explanation for the increase in endometrial and subendometrial blood flow of excessive responders by HCG +7. However, the mechanisms and control of angiogenesis in the endometrium are far from being fully understood (Smith, 2001Go).

This study is limited by the small number of patients, as only patients without ET were recruited because of concerns of power Doppler ultrasound on subsequent fetal development (Hershkovitz et al., 2002Go). Endometrial and subendometrial blood flow was measured during the early luteal phase and the observations could be extended to the later part of the luteal phase, although changes during that period may reflect preparation for menstrual bleeding. Doppler flow measurement of spiral vessels was not performed in this study because of inconsistent waveforms obtained from colour Doppler signals of these tiny spiral vessels. Doppler flow indices of spiral arteries are not predictive of pregnancy (Zaidi et al., 1995Go; Yuval et al., 1999Go; Schild et al., 2001Go), although Battaglia et al. (1997Go) and Kupesic et al. (2001Go) found significantly lower spiral artery PI in pregnant cycles than non-pregnant cycles. There is no standard definition of the subendometrial region in the literature, ranging from 5 mm (Schild et al., 2001Go; Wu et al., 2003Go) to 10 mm (Chien et al., 2002Go). Others did not specify the subendometrial region (Zaidi et al., 1995Go; Battaglia et al., 1997Go; Maugey-Laulon et al., 2002Go). In this study, we chose 1 mm of the originally myometrial–endometrial interface as the subendometrial region because the subendometrial region may extend beyond the uterine contour especially in the corneal region if 5 mm is taken. Fibroids, if present, would also not be included when 1 mm was used. Moreover, only the myometrium immediately underlying the endometrium exhibits a cyclic pattern of steroid receptor expression similar to that of the endometrium (Noe et al., 1999Go).

In conclusion, changes in uterine Doppler flow indices, and endometrial and subendometrial 3D power Doppler flow indices during the early luteal phase were significantly different between moderate and excessive responders. Larger studies with extension of ultrasound examinations to the later part of the luteal phase are needed to confirm the findings of this preliminary study.


    Acknowledgements
 
This study was funded by the Hong Kong Research Grant Council (HKU 7280/01M).


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 Materials and methods
 Results
 Discussion
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Submitted on December 12, 2003; accepted on February 11, 2004.