1 Department of Obstetrics and Gynaecology and 2 Department of Medical Physics and Bioengineering, University Hospital of Wales, Heath Park, Cardiff CF4 4XN, UK
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Abstract |
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Key words: follicles /IVF /power Doppler /ultrasonography /vascularity
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Introduction |
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The female reproductive system (ovary, uterus and placenta) contains some of the only adult tissues in which angiogenesis occurs as a normal process (Bassett, 1943; Hudlicka, 1984
; Findlay, 1986
; Ryan and Makris, 1987
; Koos, 1989
; Klagsbrun et al., 1991). This process results in rapid, periodic growth and subsequent regression of tissue accompanied by rapid changes in blood flow (Rosenfeld et al., 1974
; Koos, 1989
). The changes in blood flow in both large and small vessels, and the differences in blood flow between pregnant and non-pregnant cycles, can be monitored using colour Doppler imaging (Reynolds, 1986
; Collins et al., 1991
; Merce et al., 1992
; Tekay et al., 1995
). It has been suggested that the measurement of uterine artery pulstility index (PI) (<l 3.0) on the day of embryo transfer could predict conception in patients undergoing IVF and embryo transfer cycles (Steer et al., 1992
). More recently, Oyesanya et al. (1996) derived a vascularity index based on the number of follicles with detectable flow velocity waveforms divided by the total number of follicles in ovaries containing a minimum of 20 follicles, and this appeared to correlate with the oocyte recovery rate from patients undergoing IVF.
Power Doppler imaging (PDI) is a relatively new mode of Doppler ultrasonography which is more sensitive than conventional colour Doppler imaging (CDI) at detecting low velocity flow and hence improves the visualization of small vessels (Rubin et al., 1994; MacSweeney et al., 1996
). Chui et al. (1997) evaluated perifollicular vascularity using PDI and a subjective grading system. They found a trend towards higher fertilization rates, significantly lower triploidy rates and higher pregnancy rates when oocytes were obtained from follicles with higher grade vascularity, suggesting that perifollicular vascularity may be a predictor of treatment outcome. The purpose of the present study was to evaluate PDI assessment of perifollicular vascularity and compare it with number of outcome parameters in a series of 200 IVF and embryo transfer cycles. Follicle vascularity on the day of oocyte retrieval was divided into two groups, high (grades 3 and 4) or low (grades 1 and 2).
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Materials and methods |
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The stimulation protocol adopted in our unit has been described elsewhere (Chui et al., 1997). This consisted of menstrual delay using 5 mg twice daily of norethisterone from day 21 of the preceding cycle until 2 days prior to the predetermined day 1 of the treatment cycle. Gonadotrophin releasing hormone (GnRH) analogue, buserelin (Suprefact; Hoechst UK Ltd, Hounslow, UK) 250500 µg (0.250.5 ml) was administered s.c. either as an ultrashort protocol on days 24 or as a short protocol from day 2 until the day of HCG administration. Women with a history of polycystic ovarian disease or endometriosis received long pituitary down-regulation using a GnRH analogue prior to the commencement of ovarian stimulation. The starting dose for stimulation was 225 IU of human menopausal gonadotrophin (Pergonal; Serono Laboratories, Welwyn Garden City, UK; or Humegon or Normegon; Organon Laboratories, Cambridge, UK) or urofollitrophin (Metrodin High Purity; Serono Laboratories, Welwyn Garden City, UK) administered daily from day 3 until an appropriate response was obtained. More recently, the stimulation protocol was replaced by 150 IU of recombinant follicle stimulating hormone (FSH) (Puregon, Organon; Gonal F, Serono). Ten thousand units of HCG (Profasi; Serono Laboratories, Woking, UK) were given i.m. when at least two follicles were
18 mm in mean diameter. Serum oestradiol and luteinizing hormone (LH) concentrations taken on the day of HCG were retrospectively reviewed in pregnant and non pregnant cycles. Transvaginal follicular aspiration was performed 3536 h after HCG administration.
On the day of oocyte retrieval, each patient underwent a single transvaginal Doppler ultrasound scan using a Toshiba 140 or 270A scanner equipped with 6 MHz curvilinear transvaginal probes. The velocity range, wall filter and colour gain were standardized for both scanners, in all scans performed. The first part of the scan involved the assessment of the PI of both uterine arteries and the intraovarian (stromal) arteries in both (if present) ovaries using pulsed Doppler and colour flow mapping. The vascularity of follicles with the most extensive perifollicular vascularity grades was studied by the authors (P.S.B., D.K.C. and N.P.) using PDI to obtain a `perfusion map' of each follicle. This is described below.
The vascularity of each follicle was subjectively graded during scanning using Power Doppler imaging. The grading system devised was based on an assessment of the percentage of the perifollicular circumference in the `perfusion map' that depicted vascularity (Chui et al., 1997) and was as follows: grade 1: <25%; grade 2:
25% to <50%; grade 3:
50% to <75%; grade 4:
75%. Grades 1 or 2 were defined as low grade vascularity and grades 3 or 4 as high grade vascularity.
The mean follicular diameter and the position within the ovary of each studied follicle was also recorded. A maximum of 10 follicles per patient were selected as `study follicles' prior to oocyte retrieval, defined as those follicles with the `best vascularity' (i.e. the most extensive perifollicular vascular perfusion). A hard copy of the print of the follicle position was obtained in order to assist in the identification of individual follicles at the time of oocyte collection.
The identification of study follicles at oocyte retrieval, embryological processing, embryo selection and transfer including the type of luteal phase support and confirmation of pregnancy have been described elsewhere (Chui et al., 1997). Early pregnancy losses included ectopic pregnancies and clinical miscarriages or missed abortions by 8 weeks gestation.
Statistical analysis
The reproducibility of interobserver vascularity assessment was analysed using kappa (k) values. The k value [± 95% confidence interval (CI)]for interobserver variation of 125 follicles using the subjective grading system, based on four grades (14) was, k = 0.81 ± 0.08. Statistical analysis was by Student's t-test on mean values and 2 on outcome rates related to vascularity grades. P < 0.05 was considered to be significant. Odds ratios (OR) with their 95% CI were also used to measure the strength of association between vascularity grades and outcome variables.
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Results |
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The mean follicular diameter (MFD) was observed to increase marginally as the vascularity grade increased (Figure 2). This marginal increase was statistically significant (P < 0.05; unpaired t-test) especially between low (MFD and 95% CI; 19.6 ± 0.4 mm) and high (MFD and 95% CI; 20.3 ± 0.3 mm) follicular vascularity grades.
Oocyte retrieval, oocyte maturity and fertilization rates were all significantly higher (P < 0.05) and triploidy rates significantly lower (P < 0.05) when high and low perifollicular vascularity grades were compared (Table III). However, embryo morphology (grade A) was not significantly different.
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Discussion |
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We developed a grading system to obtain a semiquantitative assessment of the presence of an echo signal detected by power Doppler (grades 14 vascularity grades). Despite its subjectivity, in our centre we achieved good interobserver agreement with high k values (0.730.89) which illustrates the applicability and reproducibility of our results.
The assessment of uterine and intraovarian artery circulation either on the day of HCG injection or following oocyte retrieval has been suggested as a useful marker of pregnancy potential among women undergoing IVF treatment (Barber et al., 1988; Sterzik et al., 1989
; Stronhmer et al., 1991; Steer et al., 1992
; Weiner et al., 1993
). However, this study fails to suggest any correlation between the likelihood of pregnancy and either of these pulsed Doppler parameters. Other authors have similarly shown that these parameters do not appear to reliably predict outcome (Tekay et al., 1995
; Chui et al., 1997
). These conflicting findings could be due to the different treatment protocols employed which may have varying degrees of influence on the uterine vasculature (Weiner et al., 1993
; Tekay et al., 1995
) coupled with the difficulty in measuring ovarian artery signals during transvaginal scanning (Aleem et al., 1994
). Furthermore, it is important to note the differences in the population studied in terms of day of investigation, which was cross-sectional in this study as compared to other longitudinal studies (Weiner et al., 1993
)
A potential link between follicle size and vascularity has been suggested (Balakier and Stronell, 1994). Data from this study support that finding; since the trend was for mean follicle diameter in low vascularity grade follicles to be smaller than in high grade perfusion follicles (P < 0.05). This occurred despite the standardization of both HCG administration (two or more follicles were >18 mm) and the timing of oocyte collection following HCG. One explanation could be that the coefficients of variation for inter- and intra-observer errors in relation to follicular diameter measurements in this study were 4.6% and 2.9% respectively (P.S.Bhal, personal communication). However, it may also reflect the increased maturity of high grade vascularity follicles, particularly as the selection of study follicles was independent of follicular size and based only on those with the best perfusion. Pulsed Doppler studies have suggested an increase in intrafollicular blood flow in the periovulatory period (Collins et al., 1991
; Campbell et al., 1993
) which coincides with the LH surge and oocyte maturity prior to ovulation. Blakier and Stronell (1994) also showed that the perifollicular peak systolic velocity increases, especially following the administration of HCG, with a strong positive correlation with increasing follicular size. The significance of this remains obscure (Balakier and Stronell, 1994
) although it has been postulated that changes in the periovulatory follicle and its vascularity may initiate important biochemical events within the follicular environment needed for successful reproduction (Nargund et al., 1996b
). Alternatively, low grade follicle vascularity pre-HCG may affect the uptake of HCG and result in impaired maturation of the cumulusoocytecomplex. Also, higher grade perfusion may lead to the increased access of FSH to those follicles, promoting better maturation.
The oocyte retrieval rate and proportion of mature oocytes were both significantly higher (P < 0.001) in our series as was the yield of mature oocytes (P < 0.05) from follicles with high grade vascularity. This may again reflect follicular maturity. Nargund et al. (1996a,b) showed that an increase in the peak systolic velocity in vessels supplying individual follicles immediately before follicle aspiration was significantly correlated with whether or not an oocyte was obtained. This contrasts with the data from Chui et al. (1997) which did not indicate any difference in oocyte retrieval rates related follicular vascularity. However, this difference in oocyte retrieval rates may be due to the larger sample size in the present series.
Together with the correlation between follicle size and the increased number of mature oocytes retrieved, there was a significant difference (P < 0.05) in fertilization rates. This was strongly associated (OR 2.0, CI 1.33.0) with high grade vascularity follicles and appeared to be independent of semen quality; there was no perceivable difference between semen parameters between the pregnant and non-pregnant groups (Table II). Studies have suggested that follicles yielding non-fertilizable oocytes are characterized by poor or slower growth, suggesting that follicle size may be an indicator of follicular maturity (Pelluso et al., 1990
; Wittmaack et al., 1994
). Although other authors have found a relationship between follicles with higher vascularity and embryo quality (Nargund et al., 1996b
) this was not shown in the present study.
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The difference in pregnancy rates between treatment cycles categorized by embryo transfer from high or low vascularity perfusion follicles was significant (P < 0.05), but differences in early pregnancy loss (EPL) rates were not significant (Tables IV and V). However, there were insufficient low grade vascularity pregnancies to make a valid statistical comparison. The pregnancy rates in this study differ from that of Chui et al. (1977) in that the grade 4 pregnancy rate was lower at 38%, with a tendency towards a higher grade 3 and even a lower grade 2 pregnancy rate, possibly because the present data show greater diversity and size of patient population studied. The overall trend suggests that embryos derived from higher grade vascularity follicles lead to significantly higher pregnancy rates. When the differences in early pregnancy loss were analysed on the basis of the four vascular grades (Figure 3), there was a trend to a lower EPL rate in cycles where embryos transferred were derived from follicles with higher vascularity (grade 4 versus grades 2 and 3: 24% versus 82%; P < 0.05,
2-test). This supports the hypothesis that high grade follicular blood flow may be necessary for implantation and continuation of pregnancy (Chui et al., 1997
). The mechanism underlying the link between follicular neovascularization, implantation and pregnancy outcome remains unclear at present. It has been suggested that there is an association between other follicular markers and IVF outcome (Michael et al., 1995
; Gregory and Leese, 1996
). Expression of angiogenic factors within the follicular and luteal environment has been observed in animal models (Taraska et al., 1989
; Redmer et al., 1991
). These factors may help to maintain the vasculature and well-being of the pre-ovulatory follicle and participate in the recruitment of new blood vessels in the early corpus luteum (Anderson 1926
; Taraska et al., 1989
; Redmer et al., 1991
; Zheng et al., 1993
). High grade vascularity may therefore promote viable pregnancies because of its effect on the corpus luteum.
Transvaginal power Doppler ultrasonography and perifollicular vascular assessment using our subjective grading system may be used to improve the outcome of IVF and embryo transfer treatment cycles, by identifying follicles with high grade vascularity. Perifollicular vascular perfusion could be a useful additional marker in the process of embryo selection for transfer, enabling the transfer of fewer embryos with a better potential for implantation, and reducing the incidence of multiple gestation. As the outcome parameters for low grade follicular vascularity grades are poor, the possibility of identifying such follicles prior to the administration of HCG would be helpful, allowing a more efficient selection of patients completing IVF treatments. Patients with follicles mostly of low grade vascularity (preferably pre-HCG) could be carefully counselled and then given the option to abandon that treatment cycle. Further longitudinal data would be needed before this form of prospective management of IVF treatment cycles could be applied clinically.
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Acknowledgments |
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Notes |
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References |
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Submitted on April 21, 1998; accepted on November 30, 1998.