Serial uterine artery Doppler velocity parameters and human uterine receptivity in IVF/ICSI cycles

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1 Ultrasound Obstet Gynecol 2008; 31: Published online 4 February 2008 in Wiley InterScience ( DOI: /uog.5179 Serial uterine artery Doppler velocity parameters and human uterine receptivity in IVF/ICSI cycles D.A.HOOZEMANS,R.SCHATS,N.B.LAMBALK,R.HOMBURGandP.G.A.HOMPES VU University Medical Center Amsterdam, IVF Center, Amsterdam, The Netherlands KEYWORDS: embryo implantation; ICSI; IVF; serial Doppler ultrasound ABSTRACT Objective To evaluate the predictive value of serial uterine artery Doppler ultrasound for embryo implantation in in-vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) cycles. Methods This was a prospective observational study at the VU University Medical Center, Amsterdam. Patients with an indication for IVF or IVF-ICSI according to departmental protocol underwent controlled ovarian hyperstimulation followed by IVF or IVF-ICSI and embryo transfer and had serial Doppler ultrasound performed during this treatment cycle. Patient and cycle characteristics, number of conceptions and ongoing pregnancies and pulsatility index (PI) of both uterine arteries on different cycle days were assessed and results were compared between patients who conceived and those who did not. Results Of the 102 patients enrolled into the study, 83 underwent embryo transfer. Of these, 41 became pregnant and 42 did not (Group 1). Of the 41 pregnancies, 30 were ongoing (Group 2) and 11 miscarried (Group 3). Between Groups 1, 2 and 3, linear regression revealed no significant difference between any of the variables examined except in the quality of transferred embryos. There was no significant difference in the mean PI of the left and right uterine arteries on any day of the cycle, or in the change in PI during the cycle. Receiver operating characteristics curves derived to determine the performance of PI to predict pregnancy outcome supported our findings that the uterine artery PI is not a suitable marker for identifying patients with implantation failure. Multivariate analysis showed no relationship between pregnancy and PI between groups, but it did show a relationship between pregnancy and some patient and cycle characteristics. Conclusion In an unselected group of patients undergoing IVF or IVF-ICSI and embryo transfer, serial Doppler ultrasound examination of the uterine artery does not discriminate between cycles resulting in ongoing pregnany, miscarriage and no pregnancy. Copyright 2008 ISUOG. Published by John Wiley & Sons, Ltd. INTRODUCTION The major limiting factor for success in in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment cycles is embryo implantation. Despite developments in laboratory techniques and ovarian stimulation protocols, the implantation rate per embryo transferred remains approximately 15% 1. Embryo implantation depends on two parallel and probably interacting processes: embryonic development, which seems most crucial, resulting in an embryo equipped for implantation; and endometrial development, leading to uterine receptivity. Uterine receptivity depends on many biochemical and structural changes in the endometrium during the course of the menstrual cycle 2. Research on such factors has led to the identification of a window of implantation (5 7 days postovulatory) 3,4 outside which the uterus is refractory even to an otherwise normal embryo that has implantation potential. Identification of the window of implantation and its qualitative assessment in the individual patient has major clinical implications. Suitable assessment of uterine receptivity could be achieved through ultrasound because of its noninvasive nature. Various sonographic parameters, such as endometrial echogenicity, uterine contractility and Doppler parameters, have been evaluated as markers of receptivity 5. Doppler ultrasound seems the most popular technique among clinicians because of its reproducibility. Uterine blood flow can be assessed by Doppler ultrasound in the uterine arteries and there is evidence of an association between abnormal uterine artery blood velocities Correspondence to: Dr D. A. Hoozemans, VU University Medical Center Amsterdam, IVF Center, Poli Zuid, PO BOX 7057, 1007 MB Amsterdam, The Netherlands ( da.hoozemans@vumc.nl) Accepted: 7 June 2007 Copyright 2008 ISUOG. Published by John Wiley & Sons, Ltd. ORIGINAL PAPER

2 Serial uterine artery flow velocities and IVF-ICSI 433 and infertility 5 7 ; the pulsatility index (PI) is considered best at reflecting uterine artery blood velocity parameters. Using uterine artery PI as a marker of implantation has been found successful by some authors while it has been rejected by others Modern Doppler ultrasound techniques, with high-frequency transvaginal probes and color imaging, have far more acceptable intra- and interobserver variation than were possible in the early days of ultrasound 22. The cyclical vascular changes 2 of the endometrium, with angiogenesis and vasodilatation occurring in the implantation window, suggest a time relationship with embryo implantation. Further support for this relationship derives from the observation that known histological and biochemical 12 markers of receptivity correlate with Doppler findings 23. The window of implantation is characterized by a state of increased vascular permeability, massive endometrial angiogenesis 24 and decreased vascular resistance 2. Uterine perfusion is modulated by ovarian hormones; estrogens reduce the vascular impedance both in spontaneous cycles 6,8 and in estrogen and progesterone replacement cycles 9,10. Progesterone increases this effect towards the mid-luteal phase (i.e. the window of implantation), when vascular resistance is minimal and uterine perfusion is maximal 6,11,25. Various other vascular factors, such as prostaglandins and vascular endothelial growth factor, are involved in this monthly vascular endometrial development and their absence leads to subfertility 2. The ultimate proof of this concept of the relationship between vascular changes and successful implantation is the establishment of pregnancy in relation to uterine perfusion. Many authors have addressed the subject of uterine perfusion and its correlation to receptivity. Some found a positive correlation 13 18, while others did not 19,20. However, no previous publication has used serial measurements to assess true physiological changes during a treatment cycle. This seems imperative because the range of PI values between patients, which could lead to overlapping normal and abnormal values, is well known. Previous studies have found that cut-off PI-values for implantation failure range from to ,18.Also, the day of assessment does not seem to be of major importance for the predictive value 23. Therefore, in order to study receptivity sonographically, the patient herself should be used as a reference. In this study we focused on changes in PI in the individual patient during IVF to determine whether serial PI assessments in an IVF or IVF-ICSI cycle reflect a process of endometrial vascular development that predicts the possibility of successful implantation. We hypothesized that a decrease in PI would be observed during the IVF or IVF-ICSI cycle and that this phenomenon would be more pronounced in cycles resulting in pregnancy. To identify confounding factors, such as embryo quality, number of embryos transferred, maternal age, indication for IVF and the stimulation protocol, which might affect the chance of pregnancy directly or indirectly by influencing PI, we used multivariate analysis. METHODS Inclusion and treatment Between March 2002 and December 2003, a total of 102 patients who had an indication for IVF or IVF-ICSI treatment according to the criteria of the division of reproductive medicine of the VU University Medical Center consented to participate in this study. Indications included: idiopathic infertility, either IVF or IVF-ICSI male factor sub fertility (sperm count > 1 million for IVF and < 1 million for IVF-ICSI), and external endometriosis Grade I or II. Inclusion criteria were: age < 39 years, regular menstrual cycle with two normal, nonpolycystic ovaries present at the time of enrollment, and a follicle stimulating hormone (FSH) level < 10 IU/L on cycle day 3. Patients with tubal factor infertility (after pelvic inflammatory disease or with hydrosalpinges), a history of uterine surgery and/or apparent endometrial pathology (polyps, submucous myoma or synechia) were excluded, as were patients with clinically relevant systemic diseases, such as diabetes mellitus, ulcerative colitis, Crohn s disease, connective tissue diseases or hypertension. Smoking and a body mass index > 28 kg/m 2 were also exclusion criteria. Each patient was included only once. Patients were stimulated according to our standard long agonist protocol, starting with 30 µg ethinylestradiol and 150 µg levonorgestrel (Microgynon 30, Schering, Mijdrecht, The Netherlands) administered orally on cycle day 3 and continued until cycle day 24. From cycle day 17 we started treatment with 0.1 mg triptorelin-acetate (Decapeptyl, Ferring, Hoofddorp, The Netherlands) daily until the day of ovum pick-up. After withdrawal bleeding started, controlled ovarian hyperstimulation was commenced from cycle day 3 with recombinant FSH (follitrophin beta, Puregon, Organon, OSS, The Netherlands) in an individually adjusted dose that depended on age and antral follicle count. Patients were monitored with regular transvaginal ultrasound examinations and urinary hcg IU/ampoule (Profasi, Serono, Denhaag, The Netherlands) was administered to induce final follicular maturation according to protocol 26. Transvaginal ultrasound-guided follicular aspiration was performed 36 h after hcg administration and the number of oocytes was assessed under a microscope. Fertilization was achieved by either IVF or ICSI and the number and of embryos was noted. Embryo quality was assessed by experienced IVF laboratory technicians using a score based on the number of blastomeres; an overall score of 1 4 was given based on the scoring system described by Roseboom et al. 26 and Steer et al. 27. At 2 3 days after ovum pick-up, a maximum of two embryos was placed into the uterine cavity. The luteal phase was supported by 100 mg Progestan (Organon) three times daily until a pregnancy test was done days after ovum pick-up. Pregnancy was the primary endpoint. Clinical pregnancy was defined by serum hcg > 50 IU/L on day post follicle aspiration, increased hcg levels 1 week after initial assessment and the appearance of a gestational

3 434 Hoozemans et al. sac on ultrasound. Ongoing pregnancy was defined by positive fetal cardiac activity at 12 weeks gestation. Ultrasound Transvaginal sonographic measurements were performed by two observers (R.S. and D.A.H.). Uterine artery Doppler flow velocity measurements were carried out with the patient in a standard lithotomy position, using an Aloka SSD 4000 Prosound Pure HD ultrasound machine (Aloka, Tokyo, Japan) equipped with a 7.5- MHz transvaginal probe. The observations were made on specific cycle days as indicated in Table 1 and were carried out on mornings to avoid fluctuations due to the normal circadian rhythm of uterine blood flow. We assessed the PI after validation of the inter- and intraobserver variation (intraclass correlation coefficient (ICC), 0.92 (95% CI, ) for intraobserver variation and ICC, 0.95 (95%CI, ) for interobserver variation). The mean PI of left and right uterine arteries was used for analysis. Statistical analysis Univariate analysis was performed on the PI on the different assessment days, comparing absolute PI values in relation to pregnancy outcome. Univariate analysis was also used to relate the changes in PI values in the different outcome groups. Multivariate analysis (logistic regression model) was used to investigate the influence of possible confounders on the chances of pregnancy, using PI, maternal age, endometrial thickness on day of hcg injection, treatment (IVF/ICSI), IVF indication, primary or secondary infertility and total embryo quality as explanatory variables and pregnancy as the primary outcome variable. The power calculation indicated a sample size of 100 (alpha 5, estimated averages, 2.00 for pregnant and 3.00 for non-pregnant and estimated 75% non-pregnant and 25% pregnant). The test characteristics of uterine artery PI on different cycle days for embryo implantation were expressed by receiver operating characteristics (ROC) curves and the areas under the curves (AUC). The study was conducted according to the declaration of Helsinki revised in 1983 and in accordance with the research guidelines of our institute. The study was approved by the institute s review board and patients gave their written informed consent. RESULTS One hundred and two patients were enrolled into the study. Seventeen patient cycles were cancelled because of low response, prevention of ovarian hyperstimulation syndrome or fertilization failure. One patient withdrew her informed consent before starting ovarian hyperstimulation. One patient had to be excluded from the study due to violation of the protocol (it was later discovered that she smoked). Thus, 83 patients underwent embryo transfer of at least one embryo. Of these, 42 patients had a negative pregnancy test 15 days after ovum pick-up (Group 1), and 41 patients conceived. In the 41 pregnant patients there were 30 ongoing pregnancies, determined by ultrasound at 12 weeks gestation (Group 2), and 11 miscarriages/biochemical pregnancies (Group 3). The miscarriages, of which there were four, occurred at 7, 7, 8 and 9 weeks. Because of their low number they were not evaluated separately. It is possible, however, that these had a different etiology from the biochemical pregnancy losses, miscarriages being caused probably by embryonic factors (chromosomal abnormalities) and biochemical pregnancy losses having a more endometrial-driven etiology. Table 2 shows the patient characteristics of the participants and their treatment cycle characteristics. There were no significant differences between pregnant and non-pregnant patients with respect to age, cause of infertility, primary or secondary infertility or number of previous IVF attempts. The multivariate model revealed a correlation between pregnancy and: age (inverse correlation) and number and quality of embryos (positive correlation) and subfertility factor (data not shown). The individual treatment cycle characteristics did not yield discriminating factors for successful implantation. The only significant factor found was the embryo quality as assessed by light microscopy. In all patient groups there was a decrease in PI towards the mid-luteal phase, indicating a decrease in resistance of the uterine circulation, which would possibly allow better perfusion of the uterus (Table 3). However, there was an increase in PI at 15 days after ovum pick-up in Table 1 Timing of pulsatility index measurements during in-vitro fertilization/intracytoplasmic sperm injection treatment cycle, with corresponding physiological stages Phase of treatment cycle Cycle day (mean (range)) Event Start down-regulation cycle 3 (1 4) Start (basal value) Day of hcg administration 12 (10 14) Maximum E2 levels, pre-lh surge Day of ovum pick-up 14 (12 16) Ovulation, start luteal phase Day of embryo transfer 17 (14 19) Embryo enters uterus and process of hatching is initiated Ovulation day (20 25) Onset of nidation and trophoblast invasion Day of pregnancy test 29 (27 33) Implantation complete E2, estradiol; hcg, human chorionic gonadotropin; LH, luteinizing hormone.

4 Serial uterine artery flow velocities and IVF-ICSI 435 Table 2 Patient and in-vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) treatment cycle characteristics according to study group Group Variable 1: not pregnant (n = 42) 2: ongoing pregnancy (n = 30) 3: biochemical pregnancy (n = 7)/ miscarriage (n = 4) Age (years) 33.5 ± ± ± 4.0 Idiopathic factor 13 (31) 8 (27) 3 (27) Male factor IVF 16 (38) 12 (40) 1 (9) Male factor ICSI 12 (29) 8 (27) 5 (45) Endometriosis Grade I or II 1 (2) 2 (7) 2 (18) Primary infertility 32 (76) 20 (67) 8 (73) Secondary infertility 10 (24) 10 (33) 3 (27) Number of previous cycles 4 ± ± ± 1.64 Total FSH dose received (IU) 2428 ± ± ± 643 Number of stimulation days 12.2 ± ± ± 1.35 Number of oocytes retrieved ± ± ± 6.87 Number of embryos formed 7.45 ± ± ± 5.11 Score of transfer embryos 4.36 ± 1.14* 5.1 ± 1* 4.3 ± 1.62* Timing of embryo transfer: 2 days after ovum pick-up 7 (17) 3 (10) 1 (9) 3 days after ovum pick-up 35 (83) 27 (90) 10 (91) Endometrial thickness (mm) 9.95 ± ± ± 6 Values are mean ± SD or n (%). ANOVA or Chi-square tests were used as applicable. *P < 5. FSH, follicle stimulating hormone. Table 3 Mean (SD) pulsatility index (PI) values in different study groups undergoing in-vitro fertilization/intracytoplasmic sperm injection Group Timing of PI measurement 1: not pregnant (n = 42) 2: ongoing pregnancy (n = 30) 3: biochemical pregnancy/ miscarriage (n = 11) P Start down-regulation cycle 2.18 (7) 2.32 (0.50) 2.18 (8) 0.51 Day of hcg administration 2.11 (7) 2.19 (0.56) 2.15 (0.38) 0.78 Day of ovum pick-up 2.08 (0.38) 2.13 (0.38) 2.15 (1) 2 Day of embryo transfer 1.84 (0) 2.04 (0.59) 1.98 (0.37) 0 Ovulation day (3) 1.75 (1) 1.73 (0.36) 0.94 Day of pregnancy test 1.89 (7) 1.85 (0) 1.87 (0.50) 0.94 hcg, human chorionic gonadotropin. pregnant as well as in non-pregnant patients. There was no significant difference between the groups in the change (decrease) in PI from the start of the cycle towards the day of embryo transfer and beyond (Table 4). Table 5 presents the results of the multivariate logistic regression analysis. Odds ratios for PI were not significant, indicating that the amount of decrease in PI did not discriminate between cycles that resulted in pregnancy and cycles that did not. This was regardless of the other variables in the model. ROC curves on different cycle days indicated the poor test characteristics (sensitivity and specificity) of PI as an implantation marker (Figure 1), with areas under the curve of between 1 and DISCUSSION The success of human embryo implantation depends on maternal and embryonic factors and their interactions. To assess uterine receptivity one must take various factors into account. The main factor determining success, identified by various studies (reviewed by Hoozemans et al. 2 ), is probably embryo quality as determined by light microscopy. Our results confirmed this. We tried to increase the sensitivity of PI for implantation by performing serial measurements in the same patient, to assess true physiological changes involved in the endometrial vascular development cycle. Because vascular development is imperative for a receptive endometrium, its assessment by Doppler ultrasound might be predictive for implantation. We must now reject this hypothesis because there were no significant differences in PI on any of the cycle days and in the change in PI through the treatment cycle. We observed a wide range of PI values that were distributed almost equally between pregnant patients, non-pregnant patients and patients with a failed pregnancy (Tables 3 and 4). It is remarkable that in our

5 436 Hoozemans et al. Table 4 Mean (SD) changes in pulsatility index (PI) values between cycle days in different study groups undergoing in-vitro fertilization/intracytoplasmic sperm injection Group Period over which PI changed 1: not pregnant (n = 42) 2: ongoing pregnancy (n = 30) 3: biochemical pregnancy/ miscarriage (n = 11) P Baseline: start of down-regulation cycle 2.18 (7) 2.32 (0.50) 2.18 (8) 0.51 To day of hcg administration 8 (0.52) 0.14 (9) 4 (0.57) 4 To day of ovum pick-up 0.11 (7) 0 (7) 4 (0.57) 8 To day of embryo transfer 0.35 (0.51) 0.30 (0.78) 0.31 (0.70) 2 To ovulation day (0.53) 0.55 (0.56) 6 (7) 0.79 To day of pregnancy test 0.30 (0.59) 7 (3) 0.32 (0.96) 0.55 Values were considered significant if P < 5. (a) (b) (c) (d) (e) (f) Figure 1 Receiver operating characteristics curves for uterine artery pulsatility index measurements during in-vitro fertilization-intracytoplasmic sperm injection treatment cycles, with assessment: (a) on day of start of the cycle (area under the curve (AUC) = 1), (b) at end of stimulation phase (AUC = 3), (c) at ovum pick-up (AUC = 4), (d) at embryo transfer (AUC = 1), (e) at mid-luteal phase (AUC = 6) and (f) on day of pregnancy test, days after ovum pick-up (AUC = 0.53). univariate analysis the only significant difference in patient characteristics or cycle characteristics between pregnant patients and non-pregnant patients was embryo score of the transferred embryos. It is well known that female age, for example, is a strong determining factor in IVF success rates, but we could not substantiate this, probably due to the small size of our study group. The small size of our study group might also be responsible for the absence of significant differences in uterine vascular impedance values. Another possible explanation is that, although vascular endometrial development is crucial for implantation, it is not so crucial in the stage that we assessed. This might be because during the window of implantation the embryo is

6 Serial uterine artery flow velocities and IVF-ICSI 437 Table 5 Results of logistic regression analysis Variable Odds ratio 95% CI Change in PI to: Day of hcg administration Day of ovum pick-up Day of embryo transfer Ovulation day Day of pregnancy test Odds ratio was considered significant if a value of 1 did not fall within the 95% CI. In the model, pregnancy was the outcome and pulsatility index (PI), age, endometrial thickness on day of human chorionic gonadotropin (hcg) injection, treatment (in-vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI)), IVF indication, primary or secondary infertility and total embryo quality were explanatory variables. a known anaerobic organism which is best cultured invitro in a low-oxygen atmosphere 28. Whether/when this state changes in the intrauterine phase of treatment is not known. In fact, we did see in follow-up studies of these patients that PIs remained low (or even tended to decrease a little further) in the first trimester of pregnancy (data not shown). Around the time of hatching and attachment of the embryo, the local circumstances in the uterus are optimal, but it seems that to a great extent factors involving the embryo itself determine whether implantation will take place. In fact, the uterus is perhaps more limiting than it is facilitating to implantation. It is well known that endometrium is not essential for embryo implantation, as proved by the occurrence of ectopic pregnancies 29.The uterus is actually the only female organ that protects the mother actively from the invasive growth and potentially destructive trophoblast. One final possible explanation of our findings, with the uterine artery being some distance from the site of implantation and not supplying the endometrium exclusively, is that the assessment of uterine artery PI was not sufficiently sensitive to assess the microvascular events of embryo implantation. In conclusion, we have shown that there does not seem to be a place for PI assessment as a routine non-invasive marker of implantation. Future research should always compensate for known confounders and assess whether there is a role for PI in a more selected population, looking in more detail in and around the window of implantation. Investigation of the vascular process of embryo implantation by assessing known markers of angiogenesis and vascular growth factors combined with ultrasound will hopefully lead to greater understanding of this process. REFERENCES 1. 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