No evidence of increased uterine vascular impedance with patient ageing following IVF

J.H. Check1, C. Dietterich, D. Lurie and A. Nazari

The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School at Camden, Cooper Hospital/University Medical Center, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Camden, New Jersey, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A study was conducted to determine if uterine vascular impedance increases with advancing age in women undergoing ovarian stimulation for IVF. A group of 162 women who had undergone conventional IVF and embryo transfer was subdivided into three subgroups by age: 30–34 years (n = 49), 35–39 years (n = 79) and 40–44 years (n = 34). The pulsatility index (PI) and resistance index (RI) of the right and left uterine arteries were measured at baseline, on the day of oocyte retrieval, and at the mid-luteal phase. There were no differences in the average PI or RI by age at any phase of the cycle. In all age groups, the luteal phase values of PI and RI were lower than those observed earlier in the cycle. Endometrial thickness was not associated with age. There was no significant correlation between PI, RI and serum oestradiol and progesterone concentrations at any phase of the cycle. Ovarian response, as measured by average number of oocytes retrieved, decreased significantly with age. Thus, reduced pregnancy rates in older women undergoing ovarian stimulation are more likely to occur because of oocyte quality and quantity rather than uterine senescence. If future studies determine that the use of ovarian stimulation does decrease implantation rates more in older versus younger women, a mechanism other than increased uterine vascular impedance must be sought.

Key words: Doppler imaging/endometrial receptivity/uterine blood flow/uterine senescence


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Many IVF centres have concluded, based on the high pregnancy rates (PR) in older patients receiving oocytes donated from younger women, that the main reason for decline of fecundity with advancing age are degenerative changes within the ageing ovary rather than senescent changes in the uterus (Rotsztejn and Asch, 1991Go; Sauer et al., 1992Go; Abdalla et al., 1993Go; Balmaceda et al., 1994Go; Check et al., 1994Go; Navot et al., 1994Go; Legro et al., 1995Go; Lydic et al., 1996Go; Remohi et al., 1997Go; Sauer, 1997Go; Stolwijk et al., 1997Go). However, some donor oocyte studies suggest that there still may be a smaller adverse effect of the ageing uterus on successful implantation (Check et al., 1993aGo; Flamigni et al., 1993Go; Yaron et al., 1993Go; Cano et al., 1995Go; Rosenwaks et al., 1995Go; Borini et al., 1996Go; Levran et al., 1996Go).

There was no difference in endometrial characteristics according to age in women placed on an artificial oestrogen/progesterone regimen for the purpose of receiving fertilized donor oocytes (Sauer et al., 1993Go). The evaluation included histological changes as shown by endometrial biopsy, sonographic criteria of endometrial thickness and echo patterns, and progesterone receptor characteristics (Sauer et al., 1993Go).

The effect of advancing age on the uterus was studied by transvaginal colour Doppler sonography, and it was determined that there are changes in the flow velocity patterns of the ovarian, uterine, radial and spiral arteries with age (Kurjak and Kupesic, 1995Go). However, it was suggested by these authors that since the uterine artery resistance index (RI) does not change significantly in the first postmenopausal years, these data strongly support the hypothesis that the ageing process initially affects the uterus less than the ovary (Kurjak and Kupesic, 1995Go). A subsequent study (Cacciatore and Tiitinen, 1996Go) found that uterine artery pulsatility index (PI) was significantly lower in patients undergoing ovarian stimulation for IVF than in women evaluated in natural cycles. Nevertheless, the percentage reduction was correlated with age (Cacciatore and Tiitinen, 1996Go). Results of another study showed that age does not affect uterine resistance to vascular flow in patients undergoing oocyte donation (Guanes et al., 1996Go).

The present study evaluated a group which was four times the size of that evaluated earlier (Cacciatore and Tiitinen, 1996Go). Furthermore, the latter authors performed their study on the day of embryo transfer. Since embryo freezing seems to be more efficacious at the two pronuclear (2 PN) stage than for the 3-day-old embryo (at least using the simplified freezing and thawing protocol that we employ) (Baker et al., 1997Go), our study focused on the uterine blood flow measurement as shown by a decrease in vascular impedance on the day of oocyte retrieval. This would allow the deferment of embryo transfer if inadequate uterine measurements were obtained. Furthermore, results were obtained in mid-luteal phase, which allowed the supplementation of extra progesterone at this time if an inadequate decrease in uterine vascular resistance was found to be correlated with poor outcome. Since this study investigated uterine vascular impedance following ovarian stimulation, the findings would not necessarily apply to the uterus of an older woman trying to conceive naturally. Data exist which conclude that the median PI is significantly higher in natural cycles than cycles using ovarian stimulation for IVF (Tekay et al., 1996aGo).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study group consisted of 162 women undergoing IVF and embryo transfer between September 1, 1995 and July 31, 1997 at the Cooper Institute for IVF. Women between the ages of 30 and 44 years who had Doppler sonography performed three times during the IVF cycles were enrolled in the study. The women all underwent ovarian stimulation before oocyte retrieval. The ovarian stimulation used was either luteal phase leuprolide acetate/human menopausal gonadotrophin (HMG) (Meldrum et al., 1989Go), flare (follicular phase leuprolide acetate/HMG) (Garcia et al., 1990Go) or clomiphene citrate/HMG (Shanis et al., 1995Go). Previous studies (Choe et al., 1997Go) at our centre demonstrated that vascular impedance indices did not differ by stimulation protocol; therefore ovarian stimulation protocol was not used as a confounding variable. The women were placed into three groups based on the woman's age at the time of cycle: 30–34 years (n = 49); 35–39 years (n = 79); and 40–44 years (n = 34).

Doppler sonography with colour flow imaging to obtain measurements of the PI and RI, as well as endometrial thickness, were performed on day 2 of the cycle (flare and clomiphene citrate/HMG cycles) or at the time of down-regulation (luteal leuprolide acetate/HMG cycles) to obtain baseline parameters. These measurements were repeated on the day of oocyte retrieval and again in the mid-luteal phase. The studies were performed in the morning between 08:00 and 10:00 hours. We were at the time unaware of previous data concerning standing and uterine blood flow, so that no set time of resting before scanning was made (Dickey et al., 1994Go; Zaidi et al., 1995Go). It was estimated that, on average, each woman was lying on the table for ~5 min before Doppler sonography was performed. Patients included in the study were required to have measurements in all three phases; therefore only patients in whom embryo transfer occurred were included.

All sonographic examinations were performed on a GE Logic 400 (General Electric Corp., Milwaukee, WI, USA) using a 6.5 MHz endovaginal probe with colour and Doppler capabilities. Colour Doppler signals were obtained from the right and left uterine arteries lateral to the cervix at the level of the internal os. A Doppler range gate was then placed over each artery to obtain velocity waveforms. Recordings of each artery were considered satisfactory once multiple consecutive waveforms of equal intensity were obtained. Measurements of the PI and RI were obtained by electronically tracing the waveform. The average measurement of the right and left uterine arteries was used in the analysis.

In addition, endometrial thickness was measured by placing electronic callipers on the outer walls of the endometrium as seen in the longitudinal axis of the uterine body (Smith et al., 1984Go; Gonen and Casper, 1990Go; Check et al., 1993bGo).

Serum concentrations of progesterone and oestradiol were measured at the same three phases of the cycle: baseline, day of human chorionic gonadotrophin (HCG) treatment, and mid-luteal phase. The relationship between the hormone concentrations and vascular impedance indices was analysed. Other parameters measured were number of oocytes retrieved, fertilization rate and number of embryos transferred.

Statistical analyses included analysis of variance (ANOVA) and Pearson correlation coefficients. A P-value of 0.05 was used to assess statistical significance.

Since transvaginal sonography at the three phases of the cycle is part of the routine monitoring of our patients, IRB approval was not required. The Research Committee of the Cooper Institute for IVF reviewed the proposal and design of the study and guaranteed the confidentiality of patient records.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Comparisons by ANOVA of mean PI values at the three phases of the IVF cycle showed no significant differences by age. The mean (± SD) baseline PI in the three age groups of 30–34, 35–39 and 40–44 years were 2.51 ± 0.56, 2.50 ± 0.63 and 2.53 ± 0.59 respectively. On the day of retrieval, the mean PI in these age groups were 2.62 ± 0.44, 2.50 ± 0.37 and 2.60 ± 0.40 respectively. The mean PI values in the luteal phase were 2.18 ± 0.042 for the youngest women, 2.16 ± 0.43 for the medium-age group, and 2.41 ± 0.52 for the older women (Figure 1Go).



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Figure 1. A comparison of mean values of the pulsatility index in the uterine artery by age and phase of the IVF cycle (no significant differences).

 
The mean RI also did not differ by age when compared in each of the three phases of the cycle. At the beginning of the cycle, the mean (± SD) RI in the three age groups were 0.87 ± 0.05, 0.87 ± 0.06 and 0.87 ± 0.065 respectively. The corresponding mean RI values in the mid-cycle for the three groups were 0.89 ± 0.03, 0.88 ± 0.03 and 0.89 ± 0.04. In the luteal phase, the mean RI in the three age groups were 0.83 ± 0.05, 0.83 ± 0.06 and 0.86 ± 0.05 (Figure 2Go).



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Figure 2. A comparison of mean values of the resistance index in the uterine artery by age and phase of the IVF cycle (no significant differences).

 
Endometrial thickness (mm) was also similar at baseline, day of retrieval and mid-luteal phase in the three age groups: 5.72 ± 1.97, 11.84 ± 2.82 and 12.80 ± 3.76 for the younger women; 5.38 ± 1.94, 11.23 ± 2.31 and 11.19 ± 2.41 for the medium-aged group; and 5.71 ± 1.80, 11.03 ± 2.01 and 11.12 ± 2.79 for the older women (Figure 3Go).



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Figure 3. A comparison of endometrial thickness (mm) by age and phase of the IVF cycle (no significant differences).

 
As expected, fewer oocytes were retrieved in the older group (8.6 ± 4.7) compared with the two younger groups: 13.6 ± 8.3 and 13.3 ± 7.7 respectively (P < 0.05). There were, however, no differences between the three groups in either the number of embryos transferred: 3.3 ± 1.0, 3.4 ± 1.1 and 3.5 ± 1.3 respectively, or in fertilization rates (62.2 ± 21.5%, 58.0 ± 22.9% and 55.0 ± 20.4% respectively).

A summary of the serum oestradiol and progesterone concentrations at baseline, on the day of HCG and at mid-luteal phase is shown in Table IGo. The older age-group had significantly lower (P < 0.05) serum concentrations of oestradiol on the day of HCG and at mid-luteal phase compared with the younger groups. There were no differences between the three groups, however, in mean serum progesterone concentrations at any time interval. Serum hormone concentrations were not significantly correlated with sonographic measurements of PI, RI or endometrial thickness (Pearson correlation coefficient).


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Table I. Comparison of serum hormone concentrations by maternal age and stage of the ovarian stimulation cycle
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several studies have been published suggesting that failure to conceive despite IVF might be associated with increased peripheral resistance in the vascular bed of the uterine arteries (Baber et al., 1988Go; Goswamy et al., 1988Go; Deutinger et al., 1989Go; Strohmer et al., 1991Go; Steer et al., 1992Go; Favre et al., 1993Go; Weiner et al., 1993Go). Although implantation rates decrease with advancing age, the data presented here—which show no difference in RI or PI with advancing age at either time of retrieval or mid-luteal phase—suggest that the decline in fecundity is not related to increased uterine vascular impedance. Neither was endometrial thickness at each stage of cycle associated with age.

Mean serum oestradiol concentrations in the mid-luteal phase were significantly lower in the older group of patients. Higher serum oestradiol concentrations have been found to be associated with a lowering of the peripheral resistance to blood flow in uterine arteries (Goswamy and Steptoe, 1988Go; Steer et al., 1990Go; de Ziegler et al., 1991Go). However, since no significant differences in RI or PI were found in the luteal phase when comparing the older group with the two younger ones, the best explanation is that the lower oestradiol concentration at mid-luteal phase merely reflects the statistically lower numbers of mature follicles attained and oocytes retrieved.

Actually, one explanation why we failed to show any evidence of decreased uterine blood flow using colour Doppler sonography despite the well-known decreased PR in older patients may be that, in contrast to earlier data (Baber et al., 1988Go; Goswamy et al., 1988Go; Deutinger et al., 1989Go; Strohmer et al., 1991Go; Steer et al., 1992Go; Favre et al., 1993Go; Weiner et al., 1993Go), measurement of vascular impedance to the uterus was not associated with conception or non-conception (Favre et al., 1993Go; Coulam et al., 1994Go; Bassil et al., 1995Go; Tekay et al., 1995Go, 1996aGo, Tekay et al., bGo). If the colour Doppler measurements had been performed on different days of the cycle, the conclusions may have been different. Some studies have suggested that the measurement of uterine artery PI on the day of HCG (Zaidi et al., 1996Go), the day of follicular aspiration (Sterzik et al., 1989Go) or the day of embryo transfer (Steer et al., 1992Go) predicts subsequent implantation rates (Sterzik et al., 1989Go; Steer et al., 1992Go; Cacciatore et al., 1996Go; Zaidi et al., 1996Go).

Although there may be some uterine factors associated with decreased implantation rates with advancing age, these data strongly suggest that increased peripheral resistance on the vascular bed of the uterine arteries is not one of them. Actually, from donor oocyte studies it is not clear whether there are any definite adverse environmental effects of the aged uterus causing implantation failure. Indeed, there are studies suggesting a decrease in PR and/or implantation rates in donor oocyte programmes with advancing age (Check et al., 1993aGo; Flamigni et al., 1993Go; Yaron et al., 1993Go; Cano et al., 1995Go; Rosenwaks et al., 1995Go; Borini et al., 1996Go; Levran et al., 1996Go). However, many other studies have failed to demonstrate any reduction in fecundity with advancing age as long as oocytes from younger donors are used (Rotsztejn and Asch, 1991Go; Sauer et al., 1992Go; Abdalla et al., 1993Go; Balmaceda et al., 1994Go; Navot et al., 1994Go; Legro et al., 1995Go; Remohi et al., 1997Go; Stolwijk et al., 1997Go).

One explanation for why different conclusions were reached regarding whether there is or is not an increase in uterine vascular impedance with ageing may relate to whether the women were studied on hormonal replacement therapy in preparation for having donor oocytes, or whether they were studied after ovarian stimulation. An earlier study (Guanes et al., 1996Go) showed there to be no difference with the ageing uterus in donor oocyte recipients, while another study (Cacciatore and Tiitinen, 1996Go) showed the benefits of ovarian stimulation in reducing uterine vascular impedance to be less apparent in the ageing uterus. The present study also evaluated the effect of ageing on uterine vascular impedance following ovarian stimulation, but failed to show any differences in younger versus older patients, in contrast to data reported by others (Cacciatore and Tiitinen, 1996Go). No differences were found in the sonographic assessment of endometrial thickness either on the day of retrieval or in mid-luteal phase.

The possibility exists that differences in uterine vascular impedance are only evident on the day of embryo transfer (the day studied by Cacciatore and Tiitinen, 996) rather than on the day of oocyte retrieval, as in this study. However, we chose the day of retrieval because previous data suggested that improved implantation rates are correlated with reduced vascular impedance on this day (Sterzik et al., 1989Go), and this would be a more practical day to decide on whether to defer transfer in favour of cryopreservation of embryos.

A significant decrease in impedance to uterine perfusion as a result of increasing progesterone concentration has been demonstrated, especially in the late luteal phase (Bloechle et al., 1997Go). Thus, it is possible that the day of embryo transfer is a superior time for measurements of uterine vascular impedance compared with the day of oocyte retrieval, in that it may be more important to see if progesterone fails to further improve uterine blood flow. However, if this hypothesis were true, one may have expected the mid-luteal phase Doppler sonography findings in the present study to reflect less of a decrease in uterine vascular impedance in the ageing uterus, but they did not.

There is evidence that ovarian stimulation adversely affects endometrial receptivity (Paulson et al., 1990Go; Check et al., 1995Go, 1999Go). In contrast, there is Doppler sonography evidence that ovarian stimulation decreases uterine vascular impedance (Levi-Setti et al., 1995Go; Zaidi et al., 1996Go; Bloechle et al., 1997Go). Thus, there is a strong possibility that colour Doppler sonography measurement of uterine vascular impedance is not a good method for evaluating uterine receptivity. As mentioned, several studies have found colour Doppler sonography measurements of PI and RI not to be good predictors of subsequent outcome, except in a minority of extreme cases (Aytoz et al., 1997Go; Bassil et al., 1995Go; Bloechle et al., 1997Go; Salle et al., 1998Go). Therefore, it may be better to shift emphasis to the cytokine environment of the endometrium, with its effect on immune parameters and endometrial penetration by the invading cytotrophoblast.

Although increased uterine vascular impedance in donor oocyte recipients of advancing age is still debatable, the PR are still so good in this group (Sauer et al., 1992Go; Flamigni et al., 1993Go; Balmaceda et al., 1994Go; Bustillo et al., 1995Go; Check et al., 1995Go, 1999Go; Legro et al., 1995Go; Borini et al., 1996Go; Lydic et al., 1996Go; Remohi et al., 1997Go) that even if decreased uterine receptivity is found subsequently to exist, it would seem to be of only minor clinical importance. However, if the adverse effects of ovarian stimulation on implantation rates are exaggerated with advancing age, this could lead to changes in methodology to improve PR in this more difficult group. For example, if oestrogen/progesterone hormone replacement therapy creates an advantageous uterine environment for implantation whereas ovarian stimulation causes hostile conditions, it may be more advantageous to freeze all embryos in the older group and transfer later in a hormonally replaced cycle. A prospective study is now ongoing to test this hypothesis, as unpublished retrospective data suggest that there may be some merit to this approach.

The search for markers of uterine receptivity should continue in patients having ovarian stimulation with IVF in women of all ages to further improve implantation rates. However, based on the results of the present study, we do not believe that colour Doppler sonographic study of uterine vascular impedance is a particularly effective tool for diagnosing this problem in older patients having IVF and embryo transfer.


    Notes
 
1 To whom correspondence should be addressed at: 7447 Old York Road, Melrose Park, PA 19027, USA. E-mail: laurie{at}ccivf.com Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 26, 1999; accepted on April 5, 2000.