Hum. Reprod. Advance Access originally published online on September 12, 2006
Human Reproduction 2007 22(1):210-214; doi:10.1093/humrep/del362
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Follicular vascularity is not predictive of pregnancy outcome in mild controlled ovarian stimulation and IUI cycles
1 Infertility Unit, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena and 2 Università degli Studi di Milano, Milan, Italy
3 To whom correspondence should be addressed: Infertility Unit, Fondazione IRCCS Ospedale Maggiore Policlinico Mangiagalli e Regina Elena, Via M. Fanti 6, 20122 Milan, Italy. E-mail: dadosomigliana{at}yahoo.it
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Abstract |
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BACKGROUND: Although follicular vascularity has been shown to be a good indicator of oocyte quality in IVF, scant evidence is currently available on the predictive value of this variable in terms of pregnancy rate during controlled ovarian stimulation (COS) and intrauterine insemination (IUI) cycles. METHODS: Three-hundred and eighteen patients who had received mild COS underwent transvaginal ultrasound scan before performing the IUI. Using power Doppler imaging, vascularity of follicles with a mean diameter
16 mm was graded into a three grades according to the circumference of the follicle in which flow was identified. When more than one follicle was observed, grading was performed for all of them, and the highest vascularity grade was recorded. RESULTS: Clinical pregnancy rate (number/total) in the low-, medium- and high-grade vascularity groups was 14.1% (14/99), 10.0% (10/100) and 11.8% (14/119), respectively (P = 0.66). Similar results were observed when only monofollicular cycles were considered. CONCLUSIONS: Follicular vascularity does not predict the chance of pregnancy in women undergoing mild COS and IUI cycles.
Key words: clinical pregnancy/follicular vascularity/IUI/ovarian stimulation/power Doppler
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Introduction |
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A relationship between ovarian vascularity and chances of pregnancy has been reported since the early 1990s, but debate remains regarding the entity of this association and its clinical value (Scholtes et al., 1989
; Fleischer, 1991; Kupesic and Kurjak, 1993; Tekay et al., 1995; Bhal et al., 1999, 2001a, 2001b; Palomba et al., 2006). Instruments and indexes used to address this aspect have evolved over the last 15 years. Although initial reports used colour Doppler imaging, more recent studies employed power Doppler imaging, as this tool has been shown to be more sensitive. Moreover, variables used to quantify vascularity have changed from mathematical variables such as pulsatility or resistance index of the intra-ovarian or ovarian artery blood flow through semi-quantitative evaluation of follicular vascularity (Chui et al., 1997). At present, the most widely used quantification grading is based on the follicular circumference in which flow is identified (Chui et al., 1997; Bhal et al., 1999, 2001a; Coulam et al., 1999; Palomba et al., 2006).
There is cumulative evidence supporting the vision that follicular vascularity may be a good indicator of oocyte quality in an IVF context. Pregnancy rate is higher when embryos resulting from the fertilization of oocytes from better perfused follicles are transferred (Nargund et al., 1996; Bhal et al., 1999; Coulam et al., 1999; Huey et al., 1999; Borini et al., 2001; Palomba et al., 2006). Studies concerning in vivo fecundation are conversely scanty. To our knowledge, there is only one reported investigation regarding follicular vascularity in patients undergoing controlled ovarian stimulation (COS) and intrauterine insemination (IUI). That study documented that follicular vascularity may be an important factor in determining the success of the procedure (Bhal et al., 2001a). To gain insights into this poorly investigated aspect, we set up a large prospective study with the view to establishing the value of follicular vascularity in predicting treatment outcome in a clinical setting.
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Materials and methods |
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Patients referring to the Infertility Unit of the ‘Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena’ and selected for COS–IUI between January 2003 and December 2005 were considered for study entry. The main indications to the procedure were unexplained infertility and mild male infertility. Inclusion criteria were as follows: women aged <40 years, primary or secondary infertility lasting for at least 24 months, regular menstrual cycles, a BMI
30 kg/m2, day 3 serum FSH <12 IU/ml, normal uterine cavity and tubal patency assessed by hysterosalpingography and/or laparoscopy with chromosalpingography, normal semen analysis according to the World Health Organization criteria (WHO, 1999) or at least 5 x 106 motile spermatozoa after semen preparation and normal morphology 5% according to Kruger criteria, and no previous IUI. Patients with monolateral tubal occlusion were included if the patent tube looked normal at laparoscopy. Patients with minimal or mild (Stage I–II) endometriosis were eligible 6 months after an operative laparoscopy. They were included in the group of patients with unexplained infertility. Male factor subfertility was defined as basal semen analysis documenting semen concentration <20 x 106 spermatozoa per ml and/or progressive motility <25% and/or sperm morphology <9% according to Kruger criteria. Couples were recruited only for their first completed treatment cycle. Patients were excluded if cycles were cancelled and/or ovulation occurred before insemination and/or if they did not give their informed consent. The local Institution review board approved the study.
The precise protocol of COS and IUI cycles is reported in detail elsewhere (Ragni et al., 2006) and is herein only briefly described. Patients underwent a transvaginal ultrasonography on day 3 of the cycle and started the same day a therapeutic regimen with 50 IU recombinant FSH (Puregon®; Organon, Ooss, Netherlands) per day. Ovarian stimulation was monitored by serial transvaginal ultrasound scans starting from the 8th day of the cycle. Doses of recombinant FSH were progressively increased during ovarian stimulation if no dominant follicle was observed after at least 14 days of treatment. The GnRH antagonist Ganirelix (Orgalutran®; Organon) was administered at the dose of 0.25 mg per day starting the day in which a follicle 13–14 mm in mean diameter was visualized until hCG administration. A 5000 IU dose of hCG was administered when a leading follicle with a mean diameter 18 mm was visualized. Insemination was performed 34 h after hCG injection. No luteal phase supplementation was prescribed, as we did not find any benefit of this treatment in a previous study (Ragni et al., 2001). Cycles were cancelled if the total number of follicles with a mean diameter 16 mm was >3 and/or total number of follicles with a mean diameter 11 mm was >5 and/or in the absence of follicular growth. Clinical pregnancy was defined as the ultrasound visualization of at least one intrauterine gestational sack. Telephone follow-up was performed to assess the outcome of pregnancy.
Follicular vascularity was assessed by transvaginal ultrasound scan (EUB 6000, HITACHI equipped with a 6 MHz curvilinear colour Doppler probe). Evaluation was performed just before IUI as previously reported (Chui et al., 1997; Bhal et al., 2001a). All follicles with a mean diameter 16 mm were evaluated. The vascularity of each follicle was subjectively graded by the operator using power Doppler imaging. The grading system was adapted from previous studies (Chui et al., 1997; Bhal et al., 2001a) and is as follows: (i) low vascularity (LV), if follicular circumference in which flow was identified was one-third or less, (ii) medium vascularity (MV), if follicular circumference in which flow was identified was between one-third and two-third and (iii) high vascularity (HV), if follicular circumference in which flow was identified was two-third or more. When more than one follicle was observed, grading was performed for all of them, and the highest vascularity grade was recorded. Five different physicians performed the sonographic evaluation. All of them underwent a preliminary teaching period under the supervision of a single skilled operator (M.A.). Preliminary experiments showed an inter- and intra-observer variability both below 15%.
The sample size was calculated based on an expected clinical pregnancy rate per completed cycle of 12.0% (Ragni et al., 2006). We assumed as being clinically relevant a 50% increase and a 50% decrease in the rate of success in the high and low vascularity groups, respectively. On the basis of these assumptions and setting the type I and type II errors to the usual levels of 0.05 and 0.20, respectively, the number of cases to be enrolled per group was about 100 (corresponding to a total number of about 300 cases). Data were reported as mean ± SD or median (interquartile range) or number (percentage). They were compared using 
2 test, Fisher’s exact test, analysis of variance (ANOVA) and non-parametric Kruskal–Wallis test, as appropriate (SPSS/Windows 12.0, Chicago, IL, USA). The SD of a proportion was calculated applying a binomial distribution model.
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Results |
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Three hundred and eighteen patients were recruited. Distribution of cases according to the grade of follicular vascularity was as follows: low grade, n = 99 (31.4%), medium grade, n = 100 (31.4%) and high grade, n = 119 (37.4%). This figure confirmed our preliminary results suggesting that in this particular setting and using our classification, the grade of vascularity was uniformly distributed. Baseline clinical characteristics according to the grade of follicular vascularity are summarized in Table I. Duration of infertility was to some extent longer in the group with medium-grade vascularity, whereas BMI was slightly lower in high-grade vascularity group. Stimulation cycle characteristics are summarized in Table II. In this context, none of the considered variables were found to significantly differ according to study group.
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Overall, we observed 38 pregnancies (11.9% per cycle). Six of them were twins (15.8%); no high-order (
3 embryos) multiple pregnancies were observed. Number (rate) of clinical pregnancies in the low-, medium- and high-grade vascularity groups was 14 (14.1%), 10 (10.0%) and 14 (11.8%), respectively (P = 0.66). This result is illustrated in Figure 1. Twin pregnancies occurred in the three groups in 2, 1 and 3 cases, respectively (P = 0.74). To rule out the confounding effect of multifollicular cycles, we repeated the analysis considering specifically cycles with only one follicle with a mean diameter 16 mm (n = 181). Clinical pregnancy rate per cycle in the low-, medium- and high-grade vascularity groups was 14.8% (8 of 54), 9.7% (6 of 62) and 12.3% (8 of 65), respectively (P = 0.69). Twin pregnancies were observed in two cases (one in the medium and one in the high-vascularity groups). It is noteworthy that the study power is obviously reduced for this particular subanalysis. Specifically, the number of recruited patients would have allowed us to document at least a two-third increase and a two-third decrease in the rate of success in the high- and low-vascularity groups, respectively.
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Spontaneous abortion occurred in seven cases (18.4%). The frequency of this event in the low-, medium- and high-grade vascularity groups was 21.4% (3 of 14), 20.0% (2 of 10) and 14.3% (2 of 14), respectively (P = 0.88).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, follicular vascularity did not appear to predict the chance of pregnancy in women undergoing mild COS and IUI cycles. Pregnancy rate in the low-, medium- and high-grade vascularity groups was 14.1, 10.0 and 11.8%, respectively. To our knowledge, this study represents the largest series on this topic.
Baseline characteristics of the three study groups were not completely similar. Duration of infertility was shorter in the low-grade vascularity group, whereas BMI was higher in the high-grade vascularity group. It might be hypothesized that these differences may have influenced the chance of pregnancy per se, thus masking a possible independent effect of follicular vascularity. Overall, we do not believe that this bias may have played an important role. Of relevance here is that, even if statistically significant, both differences were extremely slight and thus of doubtful clinical relevance.
A further limitation of this study is related to the instrument used to assess follicular vascularity. A quantification grading of the follicular circumference in which flow is identified was employed. In this context, it has to be underlined that the main objective of this study was to test usefulness of vascularity assessment in a clinical setting. We were thus interested in an instrument that was both simple and commonly accepted. It is noteworthy that the method adopted in the present study currently represents the most widely used model to assess vascularity (Chui et al., 1997; Bhal et al., 1999, 2001a Coulam et al., 1999; Palomba et al., 2006). We cannot however exclude that different results may have emerged from the use of other variables such as velocities or impedance. A further possible limit is related to the grading system used. Chui et al. (1997) who first proposed this quantification method graded follicular vascularity differently. Specifically, these authors proposed a four-grade scale corresponding to the presence of the flow in <25% (F1), 25–50% (F2), 51–75% (F3) and >75% (F4) of follicular circumference. In our study, we used a three-grade scale (one-third or less, between one-third and two-third and two-third or more). There are two main reasons for this. First, previous studies indicated that the vast majority of cases were graded F3–F4, whereas only a minority of cases were graded F1–F2 (Chui et al., 1997; Bhal et al., 1999, 2001a; Palomba et al., 2006). This distribution may significantly limit the utility of a four-grade scale. Conversely, preliminary experiments using our classification demonstrated a more uniform distribution within the categories of follicular vascularity. This observation was confirmed by the results reported herein. Second, in a preliminary setting of experiments we found a higher inter-observer variability using the classification from Chui et al. into four grades rather than using our three-grades classification (data not shown).
Results emerging from the present study are in contrast with the previous investigation on this topic (Bhal et al., 2001a). In that previous trial, the authors recruited 182 patients and divided them into three groups according to the vascularity grade of follicles. If all follicles were highly vascularized (n = 101), pregnancy rate was 31%. Conversely, if they were all poorly vascularized (n = 21) or if a mixed pattern of vascularity was noted (n = 60), pregnancy rate was lower (0 and 18%, respectively). There are several reasons that may explain these conflicting results. First, the recruited populations differ. For instance, a noteworthy proportion of patients (27%) in the study from Bhal et al. underwent insemination using donor semen, whereas, in our series, this never occurred. Second, protocols of ovarian stimulation were remarkably different. Bhal et al. used regimens of stimulation with GnRH agonist (ultrashort or short protocols), whereas we used a protocol with GnRH antagonist. Moreover, the dose of gonadotrophin administered in the two studies was significantly different. Bhal et al. used daily doses of 100–150 IU of FSH, whereas in our study a mild protocol with 50 IU daily was prescribed. As a consequence, number of follicles developing per patient was markedly different. It is noteworthy that the same authors demonstrated that follicular vascularity is enhanced when ovarian stimulation is performed (Bhal et al., 2001b). Most cycles in our study were monofollicular, whereas the mean number of follicles per patient in the study from Bhal et al. was above three (Bhal et al., 2001a). This aspect represents strength of our study as it offers us the opportunity to perform a subgroup analysis focusing on patients with only one dominant follicle. That analysis included 181 cycles and confirms results observed in the entire cohort. Conversely, in the study from Bhal et al., monofollicular growth occurred only in 26 cycles thus limiting the possibility to draw reliable conclusions. Third, the study from Bhal et al. was performed in an experimental context, whereas we tested the significance of vascularity assessment in a clinical setting. In our study, inclusion criteria were less stringent and ecographic evaluations were performed by all physicians engaged in the IUI programme. Our results may thus better reflect the usefulness of a strategy of routine application of follicular vascularity assessment.
The observation that follicular vascularity is not predictive of success in IUI, although there is a consensus about its relevance in an IVF context (Nargund et al. 1996; Bhal et al., 1999; Coulam et al., 1999; Huey et al., 1999; Borini et al., 2001; Palomba et al., 2006), is somehow surprising. At least three reasons may be hypothesized to explain these conflicting observations. First, characteristics of patients selected for the two procedures may significantly differ. Second, the dosage of gonadotrophin used for ovarian stimulation is much higher in IVF cycles. Third, follicular vascularity may be blurred in the IUI paradigm by confounding factors affecting sperm and embryo transport, oocyte retrieval and in vivo fertilization. For instance, it may be hypothesized that mild endometriosis may negatively influence these events. Unfortunately, we were not able to perform a reliable subanalysis excluding women with endometriosis as only a minority of patients performed laparoscopy in our cohort.
In recent years, the growing epidemic of multiple pregnancies consequent to the widespread expansion of assisted reproductive technologies has been recognized as a major healthcare problem (Dickey, 2003). Epidemiological evidence also suggested that most multiple pregnancies arise from COS with or without IUI rather than from IVF techniques. This situation has called for new therapeutic strategies that may reduce this risk without significantly hampering the chances of success. In particular, two major strategies have been investigated in the context of COS and IUI: (i) modifying ovarian stimulation protocols using milder doses of gonadotrophins and (ii) identifying variables able to predict the hazard of multiple pregnancy so that cycles at risk may be cancelled or switched to IVF (Tur et al., 2005; Ragni et al., 2006). In this context, it has been suggested that follicular vascularity would represent an adjunctive instrument that may help physicians identify cycles at greatest risk of multiple birth (Bhal et al., 2001a). Unfortunately, results from the present study do not support this possibility at least in mild COS cycles. It is noteworthy that our results do not rule out that the assessment of follicular vascularity may be of help in COS cycles performed using higher doses of gonadotrophins.
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Submitted on May 13, 2006; resubmitted on July 16, 2006; accepted on August 3, 2006.
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