Evaluation of cycle-to-cycle variation of endometrial. responsiveness using transvaginal sonography in women undergoing assisted reproduction

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1 Ultrasound Obstet Gynecol 2002; 19: Evaluation of cycle-to-cycle variation of endometrial Blackwell Science, Ltd responsiveness using transvaginal sonography in women undergoing assisted reproduction G. S. BASIR*, W.-S. O, W. W. K. SO*, E. H. Y. NG* and P. C. HO* *Department of Obstetrics and Gynaecology, Department of Anatomy, Queen Mary Hospital, The University of Hong Kong, Hong Kong KEYWORDS: Cycle-to-cycle variation, In vitro fertilization, Natural cycles, Stimulation cycles, Suboptimal endometrium, Transvaginal ultrasound ABSTRACT Objectives To investigate the variation of endometrial responsiveness between cycles within the same women undergoing assisted reproduction. Methods The sonographic endometrial thickness in ovarian stimulation cycles was compared with that of subsequent natural cycles. One hundred and thirty-six ovarian stimulation cycles of in-vitro fertilization and embryo transfer were evaluated. Women who did not conceive in in-vitro fertilization cycles were subsequently seen in natural cycles (n = 97) or the next in-vitro fertilization cycle (n = 39). Based on a receiver operating characteristics (ROC) curve using endometrial thickness to predict pregnancy, the first in-vitro fertilization cycles were classified according to the endometrial thickness as optimal (> 8 mm) in 98 cycles, or suboptimal ( 8 mm) in 29 cycles. Similarly, spontaneous cycles were classified as suboptimal ( 7 mm) in 28 cycles and optimal (> 7 mm) in 69 cycles. Results The pregnancy rates were significantly lower (P < 0.05; Fisher s Exact test) in the suboptimal group in both the in-vitro fertilization and frozen embryo transfer cycles. There was a strong correlation (r 2 = 0.745) and a significant difference (P < 0.001; Wilcoxon signed rank sum test) between the endometrial thickness of stimulation and natural cycles. Conclusion It is possible to predict the occurrence of optimal or suboptimal endometrial response in natural cycles of women, after evaluation in stimulated cycles, with a high degree of reliability. Risk of implantation failure can be identified before subsequent treatment cycles and adjuvant therapeutic strategies may be planned to improve the endometrial response before embryo transfer. INTRODUCTION Many factors affect the success of assisted reproduction treatment. These include the embryo quality and the development of an adequate and receptive endometrium at the time of embryo implantation. In many in-vitro fertilization (IVF) programs, transvaginal sonography is used as a noninvasive method for evaluation of endometrial receptivity. The parameters most commonly used to assess this are endometrial thickness and echopattern. Endometrial sonographic response varies with the day of observation, the treatment regimen and differences in the endocrinological milieu. Results of some studies 1,2 have suggested that endometrial receptivity is markedly diminished in ovarian stimulation cycles and is one of the rate-limiting factors of IVF 3. However, there are insufficient data comparing the endometrial thickness of ovarian stimulation cycles with those in natural cycles in the same women. Nor is it known whether there is any correlation between the endometrial thickness in stimulation cycles with that in natural cycles within the same women. The aim of this study was to assess the correlation between the endometrial thickness in stimulation cycles with that in natural cycles and to establish the value of endometrial evaluation in a stimulation IVF cycle in predicting the endometrial response in subsequent cycles. MATERIALS AND METHODS Altogether, 272 IVF treatment cycles (175 ovarian stimulation cycles and 97 natural cycles) of infertile women undergoing assisted reproduction treatment in our IVF program at Queen Mary Hospital were evaluated. The endometrial thickness was studied in each IVF treatment cycle and women who failed to conceive were evaluated again in subsequent natural and stimulation cycles. Approval was obtained from the ethics committee of the Faculty of Medicine, The University of Hong Kong. Written consent was obtained from the patients before the commencement of the study. During the IVF treatment cycle, all women were stimulated with the standard protocol of controlled ovarian stimulation in our center 4. Gonadotrophin-releasing hormone agonist Correspondence: Dr G. S. Basir, Department of Obstetrics and Gynaecology, 6/F Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong ( gsbasir@hotmail.com) Accepted ORIGINAL PAPER

2 (GnRHa) was administered as a nasal spray (Buserelin, Hoechst, Frankfurt, Germany), starting on day 21 of the menstrual cycle preceding the IVF treatment cycle. A baseline ultrasound scan was performed on day 2 of the cycle to confirm downregulation and exclude any pelvic abnormality. Ovarian stimulation was initiated by intramuscular administration of human menopausal gonadotrophin (hmg; Pergonal, Serono Laboratories, Aubonne, Switzerland) daily. Follicular development was monitored by assay of serum estradiol (E 2 ), and sonographic examination of the follicles and the endometrium. Human chorionic gonadotrophin (hcg; Pregnyl, Organon, Holland) IU was given when the leading follicle was > 18 mm in diameter and there were at least three follicles with a mean diameter of 16 mm. Oocyte retrieval was performed 36 h after hcg, and embryo transfer 84 h thereafter. One of the authors (G.S.B.) performed all transvaginal ultrasound scans of the endometrium. An ultrasound machine (Aloka, Tokyo, Japan) equipped with a 5-MHz vaginal transducer was used. The scanning in stimulated cycles was performed on the day of hcg administration. The natural cycles were evaluated to assess the endometrial response under conditions of natural hormonal milieu. These women were asked to attend the assisted reproduction clinic about 18 days prior to the next expected menstrual period. Blood was taken daily for serum E 2 and luteinizing hormone (LH). When the serum E 2 was > 300 pmol/l, serial ultrasound scans were performed for the assessment of folliculogenesis and endometrial thickness until the LH surge (the day of LH surge was defined as the day on which the serum value was more than double the mean of the preceding values). Sonographic measurements on the day of LH surge in the natural cycle were used for analysis in this study. Endometrial thickness was measured on frozen gray-scale images. The maximum anteroposterior distance between the echogenic interfaces at the endometrial myometrial junction was measured in the plane through the central longitudinal axis of the uterine body. A maximum of three normally cleaving embryos were replaced in the uterine cavity 48 h after the retrieval. Excess good-quality embryos were frozen for subsequent transfer if the patient did not conceive in that cycle. Immediately before the transfer or cryopreservation, embryos were examined for the number/regularity of blastomeres and the degree of fragmentation. Embryos were graded according to the criteria of Veeck 5. Ninety seven natural cycles of 80 women were evaluated. Sixty-seven women who did not conceive in the IVF cycle and who had cryopreserved embryos were seen in subsequent natural cycles for replacement of frozen thawed embryos. Another 13 women who were not undergoing frozen embryo transfer (FET) consented to volunteer for a natural cycle evaluation before the commencement of their next IVF cycles. In three women there was lysis of the cryopreserved embryo during the thawing process and two women had fewer than two embryos for transfer. Only the first IVF and FET cycles with replacement into the uterine cavity of at least two normally cleaved embryos were evaluated for the outcome of pregnancy. We also repeated the analysis in 39 subsequent stimulation cycles in women who failed to conceive in the first IVF cycle and had to undergo another ovarian stimulation IVF cycle as part of their infertility treatment. Statistical analysis was carried out using a statistics computer program (SPSS for Windows package release 10.0; SPSS, Chicago, IL, USA). Wilcoxon signed rank sum test was used to estimate within-subject differences in stimulation and natural cycles. The relationship between endometrial thickness in stimulation cycles and in natural cycles was examined by linear regression analysis. Chi-squared or Fisher s Exact test was used to estimate the significance of difference between discontinuous variables. A value of P < 0.05 was considered significant. Sensitivity, specificity, and negative predictive values were also calculated. The receiver operating characteristics (ROC) curve analysis was used to determine the best cut-off value for endometrial thickness in predicting non-receptivity. A clinical pregnancy was defined as a pregnancy with sonographic evidence of intrauterine pregnancy or histological evidence of products of gestation. RESULTS One hundred and thirty-six women with regular menstrual cycles were studied prospectively in 175 ovarian stimulation cycles. These comprised of 136 first IVF cycles and 39 subsequent IVF cycles of women who failed to conceive in the first IVF cycles. The indications for IVF and embryo transfers included tubal, male or unexplained infertility. The women were aged between 27 and 39 years (mean (SD), 33 (2.7)). Among the 136 first IVF cycles, there were two cycles with free fluid in the uterine cavity, one bicornuate uterus, four cycles with axial uteri in which endometrial measurements were not available in the longitudinal plane, and two cycles with a uterine fibroid extending to the endometrium and distorting the uterine cavity, that were excluded from the study. In the remaining 127 cycles, embryo transfer (with at least two good embryos) was performed in 97 cycles. In 15 cycles, embryo transfer was deferred because of high E 2 levels and signs of ovarian hyperstimulation syndrome. In 11 cycles, embryo transfer could not be performed because of failure of fertilization. In another four cycles fewer than two embryos were available for transfer therefore these cycles were not evaluated for the outcome of pregnancy. In both the first stimulation cycles and the first FET cycles (Figure 1), a ROC curve was constructed applying values of sensitivities and specificities for different endometrial thickness in predicting pregnancy. The sum of sensitivity and specificity was found to be maximum at an endometrial thickness of 8 mm in ovarian stimulation cycles and 7 mm in FET cycles. These values were therefore taken as a cut-off for classification in ovarian stimulation and natural cycles, respectively. In ovarian stimulation cycles, the endometrial thickness was categorized as suboptimal (thickness of 8, mm, Group A) in 29 cycles and optimal (thickness of > 8 mm, Group B) in 98 cycles. Similarly, in the natural cycles, suboptimal ( 7 mm) endometrial thickness was found in 28 cycles of 23 women and optimal (> 7 mm) endometrial thickness in 69 cycles of 57 women. In the IVF cycles, the pregnancy rate per transfer in cycles with optimal endometrial response (20/ Ultrasound in Obstetrics and Gynecology 485

3 Figure 2 Correlation of endometrial thickness in ovarian stimulation and natural cycles. Correlation, r = 0.75; y = x. CI, confidence interval. Figure 1 Receiver operating characteristics curves in ovarian stimulation (a) and spontaneous cycles (b) of women undergoing assisted reproduction treatment. 71, 28%) was significantly higher (P < 0.05; Fisher s exact test) than that in cycles with suboptimal response (1/26, 3.8%). There was no significant difference in the serum E 2 levels or the duration of the stimulation phase between those with suboptimal and those with optimal endometrial thickness. Similarly in the natural cycles of frozen-thawed embryos, the pregnancy rate in cycles with optimal endometrial response (13/42, 30.9%) was significantly higher (P < 0.05; Fisher s exact test) than in cycles with a suboptimal response (1/20, 5%). All women in Group A exhibited suboptimal endometrial response in subsequent natural cycles. Ten women in Group A were reevaluated in an ensuing IVF stimulation cycle. The endometrial thickness remained suboptimal in all cycles. Therefore, in women manifesting suboptimal endometrial thickness in a stimulation cycle, the probability of the thickness remaining suboptimal subsequently was 100% in both natural cycles (95% confidence interval (CI), %) and stimulation cycles (95% CI, %). For women in Group B, 69 spontaneous cycles of 57 women were studied subsequently. The thickness remained optimal in 66 cycles of 54 women, whereas the endometrial thickness in three cycles became suboptimal ( 7 mm) in subsequent natural cycles. The probability of the endometrial response remaining optimal in natural cycles was 96% (95% CI, 88 99%). Twenty-nine women were reexamined in subsequent IVF stimulation cycles, and the endometrial thickness in all cycles remained optimal. Therefore the probability of the endometrial thickness remaining optimal in subsequent ovarian stimulation cycles was 100% (95% CI, %). There was a highly significant (P < 0.001) positive correlation (Figure 2) between the endometrial thickness on the day of hcg in ovarian stimulation cycles and the thickness on the day of LH surge in natural cycles (r 2 = 0.745). Using this technique of linear regression analysis, it is possible to predict the occurrence of optimal or suboptimal endometrial response in subsequent natural cycles after the women were evaluated in stimulation cycles, with a high degree of reliability. All natural cycles were assessed after a minimum of 2 months following the IVF study cycle. For ensuing stimulated cycles, there was a time lag of 3 6 months. The majority of our patients completed the study within 6 9 months of initiation. The endometrial thickness and serum E 2 in ovarian stimulation and natural cycles are shown in Table 1. In both the suboptimal endometrial response cycles and the optimal response cycles, the serum E 2 level and endometrial thickness in stimulated cycles were significantly greater than those in natural cycles (P < 0.05; Wilcoxon signed rank sum test). The characteristics in suboptimal and optimal endometrial cycles are shown in Table 2. There were no significant differences (P > 0.05) in the mean age of women, serum E 2 concentration, the number of embryos transferred, the number of follicles aspirated, the number of oocytes collected or the size of the largest follicle between the suboptimal and optimal cycles. 486 Ultrasound in Obstetrics and Gynecology

4 Table 1 Endometrial thickness and serum concentration of estradiol in stimulated and natural cycles Endometrial response Number of women Endometrial thickness (mm) Estradiol concentration (pmol/l) Suboptimal ( 8 mm) Stimulation (n = 23) a 7 (4 8) 8904 ( ) ( 7 mm) Natural (n = 23) b 6 (4 7) 757 ( ) Optimal (> 8 mm) Stimulation (n = 57) c 12 (9 27) 8638 ( ) (> 7 mm) Natural (n = 57) d 10 (8 16) 778 ( ) All values are in median and ranges. a vs. b and c vs. d: P < 0.05 (Wilcoxon signed rank sum test). Table 2 Characteristics in suboptimal and optimal endometrial response in ovarian stimulation cycles DISCUSSION Optimal cycles Mean (SD) (n = 127) Suboptimal cycles Mean (SD) (n = 39) Age of women (years; n = 127) 32.7 (2.32) 33.2 (2.7) Number of embryos transferred 2.6 (0.6) 2.6 (0.5) Number of follicles aspirated 12 (9.5) 13 (6.5) Number of oocytes collected 10 (7.4) 8.3 (7.8) Size of the largest follicle (mm)* 20.5 (3.2) 20.8 (3.2) *On the day of human chorionic gonadotrophin. None of the differences is statistically significant. The cardinal role of the endometrium is to prepare for the nidation of the embryo. Endometrial receptivity is a major factor in determining implantation. However, its identification is not always easy. The ideal test of endometrial response in infertility treatment would be a method of evaluating physiological normality non-invasively 6. If this is accurately assessed and if the uterine environment is analyzed as nonresponsive, it would allow either time to cryopreserve the embryos or alternative management in order to achieve a higher probability of conception. Transvaginal sonography is a non-invasive method of assessing endometrial response. Although endometrial sonographic thickness and pattern have been studied previously, there are few longitudinal studies comparing observations within the same women in ovarian stimulation and natural cycles or subsequent stimulation cycles. The present study shows a strong correlation of the endometrial thickness in an IVF treatment cycle with that in a subsequent natural cycle. Endometrium when categorized as suboptimal in a stimulated cycle fails to improve in cycles of a natural hormonal milieu, whereas-optimal endometrium is consistently associated with optimal development in subsequent natural or ovarian stimulation cycles. Furthermore, suboptimal endometrial response is associated with a significantly reduced pregnancy outcome and a high negative predictive value (96%) for implantation. Successful pregnancy by IVF is reported in the literature 7 despite an endometrial thickness of only 4 mm. However, in the present study, the clinical pregnancy rate was significantly lower in cycles with suboptimal endometrium when compared with cycles with optimal endometrial response. Sher et al. 8 reported that optimal endometrial grades (triple line pattern and 9 mm thick) in natural cycles were consistently associated with optimal grades in subsequent stimulated cycles (96%), while poor endometrial grades (echogenic and/or < 9 mm) in natural cycles improved only in 55% of cases during ensuing hyperstimulation. Hassan and Saleh 9 gave oral estriol to all women with a thin endometrium ( 7 mm in natural cycle) in subsequent stimulated cycles. An improvement was found in 62% of cases. Since all women were given estriol, it is uncertain whether the endometrium would show improvement in ensuing cycles without treatment. The present study substantiates and extends these observations. We initiated the study under stimulation and found that the suboptimal response usually recurs in subsequent cycles whether stimulated or natural. This could be due to endometrial insensitivity that results in impaired endometrial development and unresponsiveness to conventional therapy. We could not identify any particular cause for a suboptimal endometrial response in some women. However, in a number of women the suboptimal response may be associated with endometrial fibrosis, intrauterine synechiae, intrinsic uterine pathology, or advancing age 8. Other investigators 10 have suggested the presence of supraphysiological levels of hormones as a cause of diminished endometrial receptivity. No information, however, is yet available on the histological characteristics of suboptimal endometrium. Further studies on unresponsive endometrium are needed for better understanding of the pathophysiology and the association of this phenomenon with implantation failure. The present study showed a highly significant correlation between the endometrial thickness in the stimulated cycles and natural cycles. Similarly, repetitive cycles of ovarian stimulation were associated with a highly consistent endometrial thickness. The results also showed that the endometrial thickness in stimulated cycles is significantly greater than that in natural cycles. However, the persistently thin endometrium in some women is probably due to endometrial unresponsiveness because the serum estradiol levels in these women were similar to those with optimal endometrial response. The local mechanisms controlling endometrial proliferation and unresponsiveness have yet to be known. The endometrium is a major target site for ovarian hormones and is composed of Ultrasound in Obstetrics and Gynecology 487

5 heterogeneous cell types such as the stroma, luminal epithelium, and glandular epithelium that undergo continuous synchronized changes of proliferation and differentiation in response to changes in levels of circulating estrogen and progesterone 11,12. It is suggested that the endometrium must reach a certain stage of maturation and development before it can respond in an adequate way to support the nidation of an embryo. A recent study 13 showed that many of the effects of estrogen on the uterus are mediated by the estrogen receptors. Moreover, in suboptimal endometrial development, it is possible that there is a deficiency of the endometrial estrogen and progesterone receptors. Previous studies 2,14 16 provide evidence to support this hypothesis. It would be of interest to quantitatively examine these receptors and study the structural changes associated with persistently suboptimal endometrial growth. Unfortunately there is as yet no proven method that has been effective in managing women with unresponsive endometrium. In recent studies, the usage of low-dose aspirin has been shown to improve the implantation and pregnancy rate in some women. Similarly, there is also suggestive evidence that vaginal sildenafil may be of value in improving pregnancy rate 22. The data are still preliminary and further studies are needed before these forms of treatment can be recommended for women with a thin endometrium. Moreover, another important aspect is to set the threshold of endometrial thickness as suboptimal according to the experience of each IVF team. The present study provides longitudinal observational evidence and demonstrates that it is possible to predict the occurrence of optimal or suboptimal endometrial response in subsequent natural cycles of women, after they are evaluated in stimulated cycles, with a high degree of reliability. In a recent study 23, the investigators found that the treatment outcome was influenced marginally by endometrial thickness and they therefore advocated embryo transfer in women with inadequate endometrium. We aim to shift the emphasis from embryo transfer to achieving a better success rate in IVF. The understanding of a non-responsive endometrial phenomenon and its association with implantation failure is imperative prior to ensuing assisted reproduction treatment. In view of a significant correlation in the endometrial thickness between the ovarian stimulation and natural cycles, we believe that the clinician should look for underlying uterine pathology in women showing a suboptimal endometrial response, and consider adjuvant treatment strategy before embryo transfer. Early prediction of suboptimal response could prevent embryo wastage and help to curtail the financial constraints, and physical and psychological pressures associated with IVF treatment. However, larger studies to validate our preliminary data are needed before a change in the therapeutic management of infertile women with unresponsive endometrium could be recommended. ACKNOWLEDGMENTS The authors thank P.-M. Wu, senior physicist, Queen Mary Hospital, Dr W. Yeung, and all members of the reproduction team for their support. REFERENCES 1 Paulson RJ, Sauer MV, Lobo RA. Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil Steril 1990; 53: Hadi FH, Chantler E, Anderson E, Nicholson R, McClelland RA, Seif MW. Ovulation induction and endometrial steroid receptors. Hum Reprod 1994; 9: Gonen Y, Casper RF. Prediction of implantation by the sonographic appearance of the endometrium during controlled ovarian stimulation for in vitro fertilization (IVF). J Vitro Fert Embryo Transf 1990; 7: Basir GS, O WS, Ng EHY, Ho PC. 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6 21 Sher G, Feinman M, Zouves C, Knutzen V, Maassarani G, Salem R, Matzner W, Ching W, Chong P. High fecundity rates following in vitro fertilization and embryo transfer in antiphospholipid antibody seropositive women treated with heparin and aspirin. Hum Reprod 1994; 9: Sher G, Fisch JD. Vaginal sildenafil (Viagra): a preliminary report of a novel method to improve uterine artery blood flow and endometrial development in patients undergoing IVF. Hum Reprod 2000; 15: De Geyter Ch, Schmitter M, De Geyter M, Nieschlag E, Holzgreve W, Schneider HPG. Prospective evaluation of the ultrasound appearance of the endometrium in a cohort of 1186 infertile women. Fertil Steril 2000; 73: Ultrasound in Obstetrics and Gynecology 489