Quantifying the changes in endometrial vascularity throughout the normal menstrual cycle with three-dimensional power Doppler angiography

N.J. Raine-Fenning1, B.K. Campbell, N.R. Kendall, J.S. Clewes and I.R. Johnson

Academic Division of Reproductive Medicine, School of Human Development, University of Nottingham, UK

1 To whom correspondence should be addressed at: Academic Division of Reproductive Medicine, D Floor, East Block, Queens Medical Centre, Nottingham NG7 2UH, UK. e-mail: nick.fenning{at}nottingham.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: We used three-dimensional power Doppler angiography (3D-PDA) to examine the periodic changes in endometrial and subendometrial vascularity during the normal menstrual cycle in 27 women without obvious menstrual dysfunction or subfertility. METHODS: 3D-PDA was performed on alternate days from day 3 of the cycle until ovulation and then every 4 days until menses. Virtual organ computer-aided analysis and shell-imaging were used to define and to quantify the power Doppler signal within the endometrial and subendometrial regions producing indices of their relative vascularity. RESULTS: Both the endometrial and subendometrial vascularization index (VI) and vascularization flow index (VFI) increased during the proliferative phase, peaking ~3 days prior to ovulation (P < 0.001) before decreasing to a nadir 5 days post-ovulation (P < 0.001). Thereafter, both vascular indices gradually increased during the transition from early to mid-secretory phase. The flow index (FI) showed a similar pattern but with a longer nadir post-ovulation. Smoking was associated with a significantly lower VI and VFI. The FI was significantly lower in women aged ≥31 years and significantly higher in parous patients. CONCLUSIONS: Endometrial vascularity, as assessed by 3D-PDA, varies significantly during the menstrual cycle and is characterized by a pre-ovulatory peak and post-ovulatory nadir during the peri-implantation window.

Key words: endometrium/menstrual cycle/power Doppler/subendometrial vascularity/three-dimensional ultrasound


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Each month, the human endometrium undergoes a series of distinct cyclical changes in preparation to receive the developing blastocyst. Such changes necessitate well-controlled, dynamic remodelling of the endometrial microvasculature through the processes of angiogenesis and arteriogenesis (Smith, 2000Go; Rogers and Abberton, 2003Go). Dysregulation of endometrial blood flow has been associated with several menstrual disorders including dysmenorrhoea, menorrhagia, intermenstrual bleeding and endometriosis (Jaffe, 2000Go; Smith, 2001Go). An adequate blood supply also appears to be an important determinant of endometrial receptivity during assisted conception treatment (Friedler et al., 1996Go; Carbillon et al., 2001bGo), and it is possible that a cohort of women with unexplained infertility have decreased uterine and endometrial perfusion (Goswamy et al., 1988Go; Kurjak et al., 1991Go). Following implantation, the endometrial vasculature undergoes considerable change in association with the trophoblastic invasion of the spiral arteries, and suboptimal responses have been associated with recurrent miscarriage (Jirous et al., 2001Go; Habara et al., 2002Go), preterm delivery (Strigini et al., 1995Go), intrauterine growth restriction and pre-eclampsia (Kurdi et al., 1998Go; Aardema et al., 2001Go; Carbillon et al., 2001aGo). A better understanding of endometrial blood flow during the menstrual cycle is clearly of paramount importance in terms of both physiological and pathophysiological change and may permit the definition of new aetiologies or facilitate assessment of future therapeutic treatments.

Ultrasound has been used to record endometrial development non-invasively and, whilst the characteristic architectural variations in appearance and thickness have been well described (Randall et al., 1989Go; Bakos et al., 1993Go, 1994Go), there is much less information available on how endometrial blood flow changes during the menstrual cycle. Of the studies examining uterine perfusion during the menstrual cycle, the majority have applied pulsed wave Doppler and subsequent waveform analysis of gated signals returning from specific sections of the uterine vessels (Steer et al., 1990Go; Kupesic and Kurjak, 1993Go; Sladkevicius et al., 1993Go; Achiron et al., 1995Go; Bourne et al., 1996Go). These studies differ quite considerably in design both in terms of their patient populations and with respect to the vessels assessed, with most concentrating on the uterine artery rather than its downstream branches. Those studies that have reported upon blood flow within subendometrial vessels (Kupesic and Kurjak, 1993Go; Sladkevicius et al., 1993Go; Achiron et al., 1995Go; Tan et al., 1996Go) are limited by their selection of individual vessels and the assumption that they are representative of the subendometrium as a whole (Hoskins, 1990Go). This has to be questioned in the light of recent work during controlled ovarian stimulation treatment that has demonstrated differential flow rates with greater impedance to flow in spiral arteries located in the posterior aspect of the uterus (Hsieh et al., 2000Go). In addition, accurate and reliable measurement of blood flow velocities within any vessel requires angle correction and the generation of a well-defined, measurable waveform, both of which are difficult in spiral arteries due to their inherently low flow rates and tortuous nature (Nelson and Pretorius, 1988Go; Vieli, 1990Go).

Power Doppler is better suited to the study of the subendometrial vasculature as it is more sensitive to these lower velocities and is essentially angle independent (Rubin et al., 1994Go). Blood flow and vessel patterns are demonstrated by encoding the power in the Doppler signal rather than its mean frequency shift (Rubin, 1999Go). In combination with three-dimensional ultrasound, power Doppler provides a unique tool with which to examine the uterine blood supply as a whole as opposed to analysis of individual vessels or two-dimensional planes (Downey et al., 2000Go). Following data acquisition, the power Doppler signal can be quantified within any three-dimensional area, thereby permitting investigation of regional uterine blood flow. This technique has been used in the clinical setting to predict the response to controlled ovarian stimulation and subsequent outcome of assisted reproduction treatment (Schild et al., 2000Go; Kupesic et al., 2001Go; Kupesic and Kurjak, 2002Go; Wu et al., 2003Go).

Having demonstrated our own inter-observer reliability and validity of data acquisition and measurement with this technique (Raine-Fenning et al., 2002aGo), the aim of this study was to use ‘three-dimensional power Doppler angiography’ to quantify the changes in endometrial blood flow that occur throughout the menstrual cycle.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental design
This was a prospective, longitudinal observational study. Patients were asked to call on the first day of menstruation to book an appointment for day 3 of the menstrual cycle. They were then seen on an alternate day basis until the collapse of a dominant follicle was noted on ultrasound following a peak in serum LH, thereby signifying ovulation. Thereafter, patients were seen every 4 days until the next menstrual period. A transvaginal ultrasound scan was conducted and blood was taken for serology at each visit. We requested that patients serially attended at the same time of the day if at all possible.

Patient selection
The inclusion criteria were aimed at selecting a control population of women without menstrual dysfunction or obvious subfertility. Thirty women of reproductive age were recruited. The exclusion criteria were: (i) an irregular menstrual cycle; (ii) current hormonal contraception; (iii) intrauterine contraceptive device in situ; (iv) tubal sterilization; (v) a menstrual disorder necessitating any form of treatment; and (vi) history of endometriosis, infertility, pelvic inflammatory disease, recurrent miscarriage or polycystic ovarian disease

We did not specify an upper or lower age limit and did not limit the study to parous women because we wanted to investigate the effects of age and parity. In view of the evidence of a circadian rhythm in uterine blood flow (Zaidi et al., 1995Go), we attempted to keep the time of assessment similar in each woman whenever possible, and data acquisition was largely undertaken between 07.00 and 13.00 hours.

Approval was given by the local ethical committee, and subsequent recruitment was facilitated through local and regional advertisements. Patients were not financially rewarded for their participation in the study but did receive a small remuneration to cover travel and parking costs. Patients were interviewed by a clinician (N.J.R.F.) to determine their eligibility, outline the study and obtain written consent before enrolment.

Data acquisition
All data were acquired with a Voluson 530DTM machine (GE Kretz, Zipf, Austria) and a 7.5 MHz transvaginal probe. The ultrasound scans were conducted by one of two observers (N.J.R.F. and J.S.C.) whose inter-observer reliability of data acquisition had been established in preliminary work (Raine-Fenning et al., 2002aGo). Each patient was scanned in a supine position with knees flexed and hips abducted. The pelvis was examined in detail to exclude obvious ovarian or uterine pathology. Power Doppler ultrasound was then applied using pre-determined settings derived from preliminary work, and these were kept constant for every patient: pulse repetition frequency 1.0, power 4.0, colour gain 38.4, wall motion filter 75, rise 0.2, persistence 0.8, reject 82 and with the central frequency set to mid. These settings were found to offer the best compromise between small vessel detection and Doppler artefact (Raine-Fenning et al., 2002bGo). A longitudinal view of the uterus and endometrial cavity was obtained and the volume mode entered. The resultant truncated sector defining the area of interest was then moved and adjusted, and the sweep angle set to 90° to ensure that a complete uterine volume encompassing the entire subendometrium was obtained. The patient was asked to remain as still as possible and every effort was made by the ultrasonographer to limit inappropriate movements of the transducer. A three-dimensional 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. Particular attention was given to the coronal image in the C plane, specific to three-dimensional ultrasound, which provides more spatial information than the transverse or longitudinal image. If the volume was complete with no power Doppler artefact, the data set was stored to a magnetic optical disk. If there was apparent artefact, such as typical ‘flash’ artefacts seen with bowel movements or the patient coughing, the data set was reacquired until a satisfactory image was obtained.

Standardization of the ultrasound settings was ensured by using the same pre-defined probe programme without adjustment once the programme had been loaded. Prior to each acquisition, the power Doppler settings were checked to ensure they had not changed during manipulation of the volume sector, which can lead to an automatic increase or decrease in the settings with larger and smaller volumes, respectively. At the end of the scan session, the acquired volumes were reloaded from the magnetic optical disk and sent to a personal computer via a dedicated DICOM link (Digital Imaging and Communications in Medicine) (Mildenberger et al., 2002Go). 3D ViewTM software (GE Kretz) was used by the personal computer to receive and store the volume data sets and for the subsequent analysis of the endometrial data.

Data analysis
Data assessment was undertaken by one observer (J.S.C.) to limit bias. Three-dimensional endometrial volumetric and vascular measurements were undertaken with the virtual organ computer-aided analysis imaging program (VOCALTM) within 3D ViewTM. VOCAL allows the user to define the volume of interest manually with a standard computer mouse as the data set is rotated about a central axis, and we have previously described the application of this technique for endometrial volume measurements (Raine-Fenning et al., 2002cGo). For the purpose of this study, all measurements were conducted manually in plane C (coronal image) as this plane was rotated about plane A (longitudinal image) using the 9° rotation step (see Figure 1). Because the data set is rotated through 180°, the 9° rotation step makes 20 planes available to calculate each individual volume and represents the best compromise between reliability, validity and time to define the initial volume (Raine-Fenning et al., 2003Go). Once the endometrium had been defined, the power Doppler signal within it was quantified through the ‘histogram facility’, which employs specific mathematical algorithms to generate three indices of vascularity (Pairleitner et al., 1999Go). These indices are representative of either the percentage of power Doppler data within the defined volume (the VI; vascularization index), the signal intensity of the power Doppler information (the FI; flow index) or a combination of both factors (the VFI; vascular flow index) (see Table I for a full definition of each index) and have been suggested as representative of vascularity and flow intensity (Jarvela et al., 2003Go).



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Figure 1. Virtual organ computer-aided analysis (VOCALTM). This figure shows a typical multiplanar display of the uterus following three-dimensional power Doppler data acquisition. The longitudinal and transverse views are show in the upper right and left images, respectively, and the third view, the coronal plane, can be seen in the lower left image. The myometrial–endometrial border has been manually delineated, and the subendometrium defined through the process of shell-imaging, whereby a second contour or ‘shell’ is applied that exactly mirrors the originally defined surface contour. The resultant three-dimensional model can be seen in the lower left image.

 

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Table I. Power Doppler quantification
 
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. For the purpose of this study, we used shell-imaging to define a three-dimensional region within 5 mm of the originally defined myometrial–endometrial contour and then quantified the power Doppler signal within this subendometrial region (see Figure 1). This is an arbitrary distance but one that reflects the inner third of the myometrium and the region supplied by the radial arteries (Bulletti et al., 1985Go).

Having confirmed our own inter-observer reliability of both acquisition and quantification of three-dimensional power Doppler data from the endometrium (Raine-Fenning et al., 2002aGo), we acquired a single data set from each patient at every visit, and a single observer subsequently conducted two serial measurements of all resultant data sets. The mean of these two measurements is shown in the Results.

Hormonal analysis
Blood was centrifuged, and plasma was separated and stored at –20 °C until assayed. Steroid hormone measurements were made by radioimmunoassay as previously described, with minor modifications. Plasma estradiol concentrations were determined following extraction with diethyl ether (Glasier et al., 1989Go) and utilized estradiol-6-O-carboxymethyloximino-2-[125I]histamine as tracer (IM135, Amersham Pharmacia Biotech UK Ltd, Bucks, UK) with an antibody raised against estradiol-6-carboxymethyloximino-bovine serum albumin (BSA) (UCB A901 Biogenesis Ltd, Poole, UK) (Campbell et al., 1994Go). The sensitivity, and intra- and inter-assay coefficients of variation were 50 pmol/l, 10.6% and 13.5%, respectively. Plasma progesterone concentrations were determined by direct immunoassay using [125I]11-progesterone glucuronide (Amersham Pharmacia Biotech) (Yong et al., 1992Go) as label, and rabbit antiprogesterone 11-hemisuccinate BSA antiserum (SAPU R7044X) (Souza et al., 1997Go). The sensitivity, and intra- and inter-assay coefficients of variation were 175 pmol/l, 8.6% and 14.5%, respectively. Human LH was determined using reagents supplied by the National Institute of Diabetes and Digestive and Kidney diseases (NIDDK). NIDDK-hLH-I-SIAFP-1 (Potency 4500 IU/mg) was labelled with 125I using the chloramine-T method as previously described (Campbell et al., 1990Go). The antiserum used was NIDDK-anti-hLH-3, which was used according to the supplier’s instructions at a final dilution of 1:600 000. The reference preparation was LER 907, which had a potency of 277 IU/mg. The sensitivity, and intra- and inter-assay coefficients of variation were 1.3 IU/l, 6.6% and 12.8% respectively.

Statistical analysis
Statistical analysis was undertaken using SPSS version 10.1.4 using the repeated measures general linear model to determine differences with time.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of 49 patients interviewed, 37 met the recruitment criteria and entered the study. This number of patients was required to fulfil our aim to study 30 patients as, of these remaining 37 patients, seven were immediately excluded on their first ultrasound examination due to pelvic pathology including leiomoyomata (three patients), ovaries with a polycystic appearance (two patients), endometriosis and an endometrial polyp. Three further patients were lost during the study as two failed to produce a dominant follicle and one had findings consistent with luteinized unruptured follicle syndrome, resulting in a final study group of 27 patients. This group had a mean age of 31 years (range 20–42 years), a median parity of one, with nine nulliparous and 18 parous women, and contained eight current smokers. All nine nulliparous women were under 31 years of age, whilst of the 18 parous women, five were under 31 and the remaining 13 were aged 31 or more. The median duration of the menstrual cycle overall was 28 days divided equally between the follicular and luteal phases (see Table II).


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Table II. Subgroup analysis of the effect of compounding factors on endometrial and subendometrial vascularity
 
Endometrial vascularity
Both endometrial and subendometrial indices of vascularity demonstrated significant changes with time (P < 0.001) (see Figure 2). Both the VI and VFI increased from the mid-follicular phase, peaking ~3 days prior to ovulation. Thereafter, there was a decrease in both of these indices, reaching a nadir 5 days post-ovulation before a gradual increase during the transition from the early to mid-luteal phase. The FI showed a similar pattern but with a more pronounced fall in the late follicular phase and an earlier post-ovulatory nadir.



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Figure 2. Temporal variation in the three-dimensional power Doppler indices throughout the menstrual cycle relative to ovulation defined as day zero and indicated by the central vertical line. The lower darker line marked by triangular points represents data from the endometrium, and the lighter line above marked by circular points represents data from the subendometrium. The central point represents the mean value, and the error bars a single standard error.

 
Relationship to sex steroid profiles
The changes in all three vascular indices closely paralleled those seen in serum estradiol and progesterone during certain aspects of the menstrual cycle. These relationships are best appreciated graphically (see Figure 3). The initial increases in VI, FI and VFI paralleled the increase in estradiol during the follicular phase, but there was a loss of this relationship after ovulation. However, all three indices began to increase again as the serum progesterone levels increased during the luteal phase.



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Figure 3. The relationship between the subendometrial flow index and both serum estradiol (A) and serum progesterone (B) throughout the menstrual cycle. The central broken line indicates ovulation, defined as day zero.

 
Subgroup analysis
Whilst there were no differences between the groups in terms of the endometrial vascular indices, several factors were found to exert significant effects on subendometrial vascularity (see Table II). Smoking was associated with a significantly lower VI and VFI but not FI in the subendometrium. The subendometrial FI was significantly lower in the group of women aged 31 or more and significantly higher in parous patients. Of those patients that had conceived previously, all had gone on to have a live birth so we could not examine the effect of gravidity separately.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This is the first study to use three-dimensional power Doppler angiography to describe the periodic changes in endometrial and subendometrial blood flow that occur during the normal menstrual cycle. The vascular indices generated through the quantification of the power Doppler signal, thought to represent the degree of vascularity and perfusion within these three-dimensionally defined areas (Pairleitner et al., 1999Go; Jarvela et al., 2003Go), demonstrated a distinct pre-ovulatory peak and peri-implantation nadir. These findings are contrary to the current consensus derived from conventional pulsed wave Doppler studies which suggest that both uterine and endometrial blood flow increase steadily throughout the menstrual cycle, reaching a peak in the luteal phase (Goswamy and Steptoe, 1988Go; Scholtes et al., 1989Go; Battaglia et al., 1990Go; Steer et al., 1990Go; Santolaya-Forgas, 1992Go; Sladkevicius et al., 1993Go; Bourne et al., 1996Go; Tan et al., 1996Go; Zaidi, 2000Go). Whilst our results differ considerably from this pattern, they must be considered in terms of the technique applied to assess blood flow and against what is known of changes in the uterine vasculature through histological studies.

Assessment of blood flow
The majority of studies examining uterine perfusion during the normal menstrual cycle have used two-dimensional ultrasound and pulsed wave Doppler to determine blood flow velocity and impedance to flow within the uterine artery (Goswamy and Steptoe, 1988Go; Scholtes et al., 1989Go; Battaglia et al., 1990Go; Steer et al., 1990Go; Santolaya-Forgas, 1992Go; Sladkevicius et al., 1993Go; Bourne et al., 1996Go; Tan et al., 1996Go; Zaidi, 2000Go). Almost all report a gradual yet continuous increase in blood flow velocity in association with a reduction in the resistance to flow from the early follicular phase maximal at the time of implantation. Several groups also noted a temporary increase in resistance to flow in association with a concomitant reduction in velocity during the peri-ovulatory period (Scholtes et al., 1989Go; Steer et al., 1990Go; Collins et al., 1991Go; Sladkevicius et al., 1993Go) that has been attributed to a physical vascular obstruction induced by a transient increase in myometrial basal tone and uterine contractility (Lyons et al., 1991Go; de Ziegler et al., 2001Go). Myometrial contractions, however, are intermittent and occur less frequently with time from ovulation (Bulletti et al., 2000Go; de Ziegler et al., 2001Go), and are unlikely to explain the sustained fall in endometrial blood flow seen in this study.

The majority of these studies assume that blood flow within the uterine arteries is representative of regional uterine and endometrial perfusion. This is supported by studies that have looked at blood flow characteristics within vessels at the subendometrial level and report similar patterns of flow to those seen in the uterine arteries (Sladkevicius et al., 1993Go, 1994Go; Achiron et al., 1995Go; Bourne et al., 1996Go). Nevertheless, analysis is still generally restricted to a single vessel and complicated by the tortuous nature of the vessels and low velocity flow profiles. Power Doppler is more sensitive to these lower velocities and, in combination with three-dimensional ultrasound, provides information from the endometrium or subendometrial region as a whole, thereby giving an impression of perfusion. Indeed, our findings are much more in keeping with studies that have been designed specifically to examine perfusion within the human endometrium during the menstrual cycle.

Fraser et al. (1987Go) measured endometrial blood flow across the menstrual cycle by observing the clearance of radiolabelled xenon133 following its instillation into the uterine cavity. They demonstrated a similar pattern to that observed in this study, with a significant elevation in the middle to late follicular phase (days 10–12), followed by a substantial fall and a subsequent slow rise during the luteal phase (days 21–26) that was maintained until menstruation. More recently, Gannon et al. (1997Go) used an intrauterine laser Doppler technique to assess the weekly variation in red blood cell flux within the subendometrium during the menstrual cycle. They found that the mean endometrial microvascular perfusion varied throughout the cycle, with two distinct and significantly increased episodes of perfusion during the follicular and luteal phases of the cycle. Whilst they also noted the highest flow during the proliferative phase, contrary to our findings and those of Fraser et al. (1987Go), this peak (days 6–9) and the second luteal peak (days 17–22) both occurred at an earlier stage of the menstrual cycle. The discrepancies most probably reflect measurement technique, with both three-dimensional power Doppler ultrasound and xenon clearance being more representative of overall uterine and endometrial flow. Laser Doppler fluxmetry provides more information about small segments of the endometrium, as it measures the passage of red blood cells through a sphere of 1 mm diameter (Johansson et al., 1991Go; Mayrovitz, 1992Go). It may not, therefore, be truly representative of the entire endometrium, although Gannon et al. (1997Go) did show minimal regional variation throughout the uterus.

Not all data relating to changes in uterine blood flow are derived from work in humans. Animal studies also suggest that a decrease in uterine flow may occur following ovulation in cows (Ford et al., 1979Go; Waite et al., 1990Go), ewes (Greiss and Anderson, 1969Go) and sows (Ford and Christenson, 1979Go). Waite et al. (1990Go) demonstrated an increased uterine arterial smooth muscle tone and lower uterine artery flow rate in association with the reversal in estrogen to progesterone ratio that occurs during the luteal phase of the cycle following oestrus in the cow. Ford et al. (1979Go) reported the same relationship between uterine blood flow and the ratio of estradiol to progesterone during the oestrous cycle of non-pregnant cows. More recently, Zhang et al. (1995Go) used a hydrogen gas clearance technique to demonstrate that myometrial blood flow was significantly greater than endometrial flow in ovariectomized rats and that uterine blood flow increased in response to boluses of 17{beta}-estradiol.

Histological changes in uterine vascularity
The highest rate of endometrial cell proliferation does occur during the early proliferative phase of endometrial growth and is maximal around days 8–10 in the upper one-third of the functionalis layer (Ferenczy et al., 1979Go). Significant dilatation of the vessels within the subepithelial capillary plexus occurs during the post-ovulatory phase, leading to oedema appearing in the stroma at the time of the expected implantation (Peek et al., 1992Go). It is possible therefore that the power Doppler signal falls at this time as a result of an increase in the distance between individual vessels and a resultant decrease in microvessel spatial density (Gannon et al., 1997Go). However, whilst there is evidence of a reduction in endometrial capillary spatial density following ovulation, it tends to occur during the latter part of the luteal phase as the endometrium becomes progressively oedematous (Johannisson et al., 1987Go; Hourihan et al., 1991Go). Nevertheless, implantation itself is characterized by an inflammatory-like response associated with increased vascular permeability and vasodilatation, and these processes could theoretically affect the power Doppler signal. Similarly, during the late luteal phase, we may have seen an increase in the power Doppler signal due to an increase in endometrial vascular density associated with the progressive coiling of the spiral arteries or endometrial compaction characteristic of the late luteal phase (Shaw and Roche, 1980Go).

Relationship to sex steroids
Our three vascular indices closely paralleled the changes seen in serum estradiol and progesterone throughout the menstrual cycle (see Figure 3). Both endometrial and subendometrial vascularity demonstrated a significant elevation in the middle to late follicular phase as the estrogen to progesterone ratio increased. Animal work has shown that the increase in uterine blood flow during this phase is due to a vasodilatatory response that is reproduced by exogenous 17{beta}-estradiol administration via a nitric oxide-mediated mechanism (Vagnoni et al., 1998Go). This relationship was maintained thereafter, with all three indices of vascularity reaching maximum values around the time of the estradiol peak before falling in parallel with the reduction in the circulating estradiol to progesterone ratio to reach a nadir in the early post-ovulatory period. Goswamy and Steptoe (1988Go) described a similar pattern but in terms of an increase in the resistance to flow in association with this post-ovulatory fall in estradiol. Whilst other groups have described the subsequent loss of the relationship with estradiol at this point (Fraser et al., 1987Go), the indices of vascularity then began to rise again, closely following, although lagging behind, the post-ovulatory increase in progesterone. The vascular indices, however, continued to rise even as the progesterone levels fell during the late luteal phase, reaching some of the highest values throughout the whole menstrual cycle just prior to menstruation. This may simply reflect tissue density and the increased coiling of spiral arteries as discussed above, but may also relate to the increasing role of the renin–angiotensin system in menstrual regulation (Johnson, 1980Go).

Subgroup analysis
Whilst cigarette smoking is associated with endothelial dysfunction (Neunteufl et al., 2000Go) and an increase arterial wall stiffness (Caro et al., 1987Go), its effect on uterine blood flow is less clear. Different groups have suggested that smoking is associated with either an increase (Nordenvall et al., 1991Go) or a decrease (Castro et al., 1993Go) in the systolic to diastolic ratio of the uterine artery, whilst most report no effect at all (Morrow et al., 1988Go; Newnham et al., 1990Go; Bruner and Forouzan, 1991Go; Kimya et al., 1998Go). In this study, smoking was specifically associated with a reduction in the VI and VFI, but not the FI.

Contrary to this were the isolated findings of a lower FI in the group of women aged 31 or more and a higher FI in parous patients. Seventy-two percent of parous women (13 of 18) were actually aged 31 or more, suggesting that the effect of age outweighs the positive effect of parity. Whilst both findings may have been expected and can be explained physiologically, there is a surprising paucity of work addressing either issue. A reduction in uterine blood flow with age has been described, but only during the post-menopausal period where there is a clear correlation between years since the menopause and the resistance to flow within the uterine, radial and spiral arteries (Kurjak and Kupesic, 1995Go). Three-dimensional power Doppler angiography has been used to demonstrate a reduction in ovarian stromal vascularity with age in both women of reproductive age (Kupesic et al., 2003Go) and older peri-menopausal women (Pan et al., 2002Go), but there are no data on uterine vascularity addressing the effect of age or parity. The numbers involved, however, are small, and larger studies dedicated to factors that may influence endometrial blood flow are warranted.

Arguably one of the most important findings from our subgroup analysis was that these differences were observed within the subendometrium only and not within the endometrium. This serves to remind us that the myometrium and endometrium have separate and discrete vascular beds and should therefore be considered independently (Ramsey, 1977Go; Rogers and Gannon, 1981Go). Shell-imaging permits such discriminatory analysis and provides a novel technique with which to examine regional blood flow within any given organ or tissue.

This study has demonstrated distinct changes in vascularity during the normal menstrual cycle, as assessed by three-dimensional power Doppler, characterized by a pre-ovulatory peak and a post-ovulatory fall that reaches a nadir at the time of implantation. The changes parallel those seen in serum estradiol during the follicular phase and serum progesterone during the luteal phase. Indices of subendometrial vascularity are significantly lower in smokers and women aged 31 or more, but are increased in parous women. Three-dimensional power Doppler angiography offers a new tool with which to investigate endometrial perfusion and could potentially be used to define pathophysiological change associated with certain disease processes and to quantify the effect of various treatment modalities on these.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 26, 2003; accepted on October 6, 2003.