IN VITRO FERTILIZATION

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1 IN VITRO FERTILIZATION FERTILITY AND STERILITY VOL. 74, NO. 2, AUGUST 2000 Copyright 2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. New look at endometrial echogenicity: objective computer-assisted measurements predict endometrial receptivity in in vitro fertilization embryo transfer Renato Fanchin, M.D., Claudia Righini, M.D., Jean-Marc Ayoubi, M.D., François Olivennes, M.D., Dominique de Ziegler, M.D., and René Frydman, M.D. Department of Obstetrics and Gynecology and Reproductive Endocrinology, Hôpital Antoine Béclère, Clamart, France Received November 2, 1999; revised and accepted February 24, This paper received the Society for Assisted Reproductive Technology Prize at the Conjoint Annual Meeting of the American Society for Reproductive Medicine and the Canadian Fertility and Andrology Society, Toronto, Canada, September 25 0, Reprint requests: Renato Fanchin, M.D., Department of Obstetrics and Gynecology and Reproductive Endocrinology, Hôpital Antoine Béclère, 157, rue de la Porte de Trivaux, 92141, Clamart, France (FAX: ; E- mail: /00/$20.00 PII S (99) Objective: To determine whether endometrial echogenicity, assessed objectively by a computer-assisted system on the day of hcg administration, predicts endometrial receptivity in controlled ovarian hyperstimulation (COH) cycles for IVF-ET. Design: Prospective analysis. Setting: Assisted reproduction unit, Clamart, France. Patient(s): Two hundred twenty-one women (aged 8 years with a normal uterus and 2 grade A or B embryos transferred) undergoing 228 GnRH agonist and FSH/hCG cycles for IVF-ET. Intervention(s): On the day of hcg administration, uterine ultrasound scans were digitized with an image analysis system. Endometrial echogenicity was assessed as the ratio of the extent of the hyperechogenic transformation over the whole endometrial thickness. According to this, cycles were sorted arbitrarily into six groups: 0% (n 4), 1% 40% (n 7), 41% 50% (n 7), 51% 60% (n 55), 61% 70% (n 7), and 70% (n 28). Main Outcome Measure(s): Pregnancy and implantation rates. Result(s): The groups were similar in regard to population characteristics, ovarian response to COH, and embryology data. Pregnancy rates (59%, 57%, 5%, 20%, 16%, and 11%, respectively) and implantation rates (5%, 2%, 17%, 6%, 7%, and %, respectively) fell progressively and significantly from the low-echogenicity group to the high-echogenicity group. Conclusion(s): The present results confirm and extend previous observations that advanced hyperechogenic transformation of the endometrium is associated with poor IVF-ET outcome. (Fertil Steril 2000;74: by American Society for Reproductive Medicine.) Key Words: Ultrasound, echogenicity, endometrial receptivity, embryo implantation, controlled ovarian hyperstimulation, in vitro fertilization Embryo implantation is determined by multiple interactive events and depends on embryo quality and endometrial receptivity. During the last 2 decades, several developments in controlled ovarian hyperstimulation (COH), fertilization, and embryo culture techniques have led to an improvement in the number and quality of embryos available for ET. In contrast, endometrial receptivity has failed to benefit from parallel optimization, and its disorders are likely to represent an important cause of the suboptimal embryo implantation rates observed in IVF-ET. Indeed, direct assessment and control of endometrial receptivity are inherently impossible in actual ET cycles. Regulated by complex endocrine (1) and paracrine autocrine (2) interactions, endometrial receptivity may undergo intercycle and interindividual variations that prevent inadvertent extrapolation of information obtained from experimental cycles. Further, in COH, administration of exogenous gonadotropins may exert, either directly () or through its supraphysiologic effects on ovarian hormones (4, 5), unfavorable consequences on the endometrium, probably in proportion to the 274

2 doses administered and the magnitude of the ovarian response ( 5). These dilemmas have led numerous investigators to focus on noninvasive prognostic features of endometrial receptivity in actual ET cycles. As an alternative to endometrial biopsies, high-resolution transvaginal ultrasonography (US) makes it possible to monitor noninvasively histologic changes in the endometrium (6 11) through the analysis of endometrial echogenicity. During the follicular phase in natural cycles, the endometrium is hypoechogenic compared with the surrounding myometrium. After ovulation, endometrial echogenicity increases progressively, with hyperechogenic changes developing from the base toward the surface of the endometrium (11), probably as a result of rising P levels (12). Measurements of endometrial echogenicity performed during the late follicular phase of COH have long been tested in search of a prognostic factor for embryo implantation. However, different conclusions have been reached in published reports. Some investigators have ascertained that endometrial echogenic patterns in the late follicular phase predict IVF-ET outcome (1 18). Others, on the contrary, have failed to find a relation between endometrial echogenicity and implantation rates (19, 20). This controversy may be explained by operator-dependent variability, the use of arbitrary and heterogeneous classifications, and the lack of control for confounding factors (e.g., poor embryo quality and uterine cavity abnormalities) that influence the analysis of results. Hence, we examined a selected subset of IVF-ET patients to clarify whether endometrial echogenicity on the day of hcg administration might reflect endometrial receptivity. To overcome the subjectivity of US measurements, we assessed endometrial echogenicity objectively with a computer-assisted module for the analysis of US images. MATERIALS AND METHODS Patient Characteristics We studied prospectively 228 consecutive COH cycles conducted in 221 IVF-ET candidates with similar COH protocols. To limit the possibility of confounding factors in the analysis of our results, we selected only women aged 8 years whose uteri were morphologically normal, as confirmed by hysteroscopy and US (absence of fibroids, adenomyosis, or polyps), and who had at least two goodquality embryos (defined as embryos having blastomeres of uniform size and shape, ooplasm with no granularity, and a maximum fragmentation of 10%) available for ET. Women were excluded if the uterine position did not allow adequate visualization of the endometrial texture by transvaginal US. During the 2 or months before COH, all women underwent an assessment of their ovarian reserve that consisted of plasma FSH and E 2 measurements on cycle day. Clinical indications for IVF-ET were tubal abnormalities (42%), sperm abnormalities (9%), unexplained infertility (17%), and endometriosis (2%). Informed consent was obtained from all patients, and this study was approved by our internal Institutional Review Board. Protocol for COH A single injection of a timed-release GnRH agonist, triptorelin (.0 mg IM, Decapeptyl; Ibsen-Biotech, Paris, France), was administered on cycle day 2. Eighteen days later, complete pituitary desensitization was confirmed by documenting low plasma levels of E 2 ( 40 pg/ml) and LH ( 2 miu/ ml). Patients also had a conventional US examination to exclude ovarian cysts and to verify that the endometrial thickness was 5 mm. Recombinant FSH therapy (Puregon; Organon Pharmaceuticals, Saint-Denis, France) was then initiated at a dosage of 225 IU/d for the first 5 days of COH. Further FSH doses and the timing of hcg administration (10,000 IU IM, Gonadotrophine Chorionique Endo ; Organon Pharmaceuticals) were adjusted according to the usual criteria of follicular maturation determined by US and E 2 findings. Administration of hcg was performed when follicles were 17 mm in diameter and E 2 levels per mature follicle ( 17 mm in diameter) were 00 pg/ml. Oocytes were retrieved 6 hours after hcg administration by transvaginal US guided aspiration. All ETs were performed 2 days after oocyte retrieval. The luteal phase was supported with 00 mg of micronized P (Utrogestan; Besins-Iscovesco Pharmaceuticals, Paris, France) administered daily (100 mg in the morning, 200 mg in the evening) by the vaginal route starting on the evening of the day of ET. Blood Samples and Hormone Measurements In addition to routine blood sampling required for monitoring COH, all women included in the study underwent blood sampling on the day of hcg administration. Plasma FSH was measured by an immunometric technique (Amerlite kit; Ortho Clinical Diagnostics, Strasbourg, France). Intraassay and interassay coefficients of variation (CVs) were 5% and 7%, respectively, and sensitivity was 0.1 miu/ml. Plasma P was measured by RIA ( 125 I Progesterone Coatria kit; Bio-Mérieux, Paris, France). Sensitivity was 0.05 ng/ml and the intraassay and interassay CVs were 8% and 11%, respectively. Plasma E 2 was determined by an immunometric technique (Estradiol-60 Amerlite kit; Ortho Clinical Diagnostics). Sensitivity was 14 pg/ml, and intraassay and interassay CVs were 8% and 9%, respectively. Ultrasound Scans On the day of hcg administration, the women underwent US scans of a sagittal plane of the uterus with a 7.5-MHz transvaginal probe (Siemens Elegra; Siemens, Paris, France) at approximately 11:00 A.M. by a single operator. The US settings (in particular, gain and scan depth) were adjusted for optimal results. By design, the endometrium could be visualized clearly in all scans. To improve consistency, we performed measurements during the rest interval between two uterine contractions. Images were digitized and analyzed FERTILITY & STERILITY 275

3 FIGURE 1 Computer-assisted analysis of endometrial echogenicity. After digitization of uterine images, multiple transverse cuts across representative sections of the endometrial surface were performed, and gray-level analysis was displayed graphically. Endometrial echogenicity was calculated as the ratio of the extent of the hyperechogenic transformation over the whole thickness of the endometrium. The top and bottom represent the analysis of a hypoechogenic and hyperechogenic endometrium, respectively. Hyperech. hyperechogenicity; ant. anterior; post. posterior; End. endometrial. Fanchin. Endometrial echogenicity and receptivity. Fertil Steril online with the use of a computer-assisted module specifically developed for the assessment of endometrial echogenicity and thickness (IôTEC.1; IôDP, Paris, France). As illustrated in Figure 1, after digitization of the uterine images, our system performed multiple transverse cuts across representative sections of the endometrial surface. Gray-level analysis was then performed automatically in all cuts, and means of the results were displayed graphically. The magnitude of the base-to-surface expansion of endometrial hyperechogenicity was calculated as the ratio of the extent of the submyometrial hyperechogenic transformation over the whole thickness of the endometrium. From a qualitative standpoint and on the basis of our previous observations, we considered the endometrial texture by US as hyperechogenic when echogenicity values exceeded those of the surrounding myometrium by 10%. Endometrial borders were set arbitrarily as the outer limits of the hyperechogenic myometrium endometrium interfaces. Endometrial thickness (truly double endometrial thickness) was calculated as the greatest distance between the outer limits of the proximal and distal endometrial interfaces. Sensitivities of the endometrial echogenicity and thickness calculations were 0.01 (1%) and 0.1 mm, respectively. Intraanalysis CVs of our measurements were 5%. As displayed in Figure 2, to simplify the interpretation of a possible relation between the degree of endometrial echogenicity and IVF-ET outcome, and on the basis of our previous experience, we created six arbitrary groups according to the extent of hyperechogenic transformation of the endometrium over the whole endometrial thickness: 0%, 1% 40%, 41% 50%, 51% 60%, 61% 70%, and 70%. Statistics Measures of central tendency used were arithmetic means, and measures of variability were SEs. When the data distribution was nonparametric, medians and ranges were used. Statistical assessment of our results was performed with factorial analysis of variance when the data distribution was normal. The Kruskall-Wallis test was used when normality of the data could not be confirmed. The hormonal influence on endometrial echogenicity and thickness was assessed with the use of simple regression. P.05 was considered statistically significant. RESULTS Overall Data The median age of the patients was 1.8 years (range, 25 7 years), and mean ( SE) cycle-day plasma FSH and E 2 levels performed in a previous cycle were within the 276 Fanchin et al. Endometrial echogenicity and receptivity Vol. 74, No. 2, August 2000

4 FIGURE 2 The cycles were sorted into six groups according to the extent of the upward hyperechogenic transformation of the endometrium. Fanchin. Endometrial echogenicity and receptivity. Fertil Steril normal range, at miu/ml and 27 1 pg/ml, respectively. COH lasted days and required IU FSH ampules. On the day of hcg administration, plasma P and E 2 levels reached ng/ml and 2, pg/ml, respectively. Mean ( SE) numbers of mature oocytes retrieved and of available embryos were and , respectively. The median number of embryos transferred was (range, 2 4). TABLE 1 Patients, COH and embryology data in the endometrial echogenicity groups. Endometrial echogenicity 0% (n 4) 1 40% (n 7) 41 50% (n 7) 51 60% (n 55) 61 70% (n 7) 70% (n 28) Ages (y) a (25 6) (26 7) (26 7) (26 7) (25 7) (25 7) Indications Tubal 41% 2% 4% 40% 51% 46% Male 7% 4% 40% 42% 2% 5% Idiopathic 19% 22% 14% 16% 14% 15% Endometriosis % % % 2% % 4% Plasma FSH b (miu/ml) Plasma E b 2 (pg/ml) IU-FSH ampules Day of hcg injection Plasma E c 2 (pg/ml) Plasma P c Mature oocytes Available embryos Transferred embryos a Endometrial thickness (mm) Note: Differences among groups are not statistically significant. a Values are medians (ranges). b On cycle-day, within the 2 months prior to COH. c On the day of hcg administration (P, progesterone). Fanchin. Endometrial echogenicity and receptivity. Fertil Steril FERTILITY & STERILITY 277

5 FIGURE Clinical pregnancy rates according to the extent of hyperechogenic endometrial transformation assessed on the day of hcg administration (P.001). Fanchin. Endometrial echogenicity and receptivity. Fertil Steril Mean ( SE) echogenicity values were (51% of the endometrial surface) and ranged from 0.17 to 1.00 (17% 100% of the endometrial surface). As shown in Figure 2, all 228 COH cycles were sorted into 6 echogenicity groups as follows: 0% (n 4), 1% 40% (n 7), 41% 50% (n 7), 51% 60% (n 55), 61% 70% (n 7), and 70% (n 28). The overall endometrial thickness was mm (range, mm). No correlation was observed between plasma P and E 2 levels, or E 2 to P ratios, and endometrial echogenicity and thickness on the day of hcg administration. Data on Population Characteristics, COH, and Embryology for Each Echogenicity Group Table 1 summarizes the patient characteristics, COH data, and embryology data for each endometrial echogenicity group. All echogenicity groups were similar with regard to age of the patients, indications for IVF-ET, ovarian reserve assessment (cycle-day FSH and E 2 levels), number of 75-IU FSH ampules administered, duration of COH, plasma E 2 and P levels on the day of hcg administration, number of mature oocytes retrieved, and the number of available and transferred embryos. Endometrial thickness (mean SE) was comparable in all six echogenicity groups, at mm (range, mm), mm (range, mm), mm (range, mm), mm (range, mm), mm (range, mm), and mm (range, mm), respectively. Data on Pregnancy and Implantation Rates for Each Echogenicity Group In contrast to the similarity in individual, COH, and embryology data among groups, the clinical and ongoing pregnancy rates (PRs) as well as implantation rates decreased dramatically from the lowest to the highest endometrial echogenicity groups (59%, 50%, 5%; 57%, 46%, 2%; 5%, 22%, 17%; 20%, 15%, 6%; 16%, 8%, 7%, and 11%, 7%, %, respectively, in the 0%, 1% 40%, 41% 50%, 51% 60%, 61% 70%, and 70% echogenicity groups; P.001). The marked fall in clinical pregnancy rates is illustrated in Figure. Conversely, no relation was observed between endometrial thickness on the day of hcg administration and IVF-ET outcome. DISCUSSION The present study investigated possible relations between the magnitude of the hyperechogenic transformation of the endometrium assessed on the day of hcg administration and IVF-ET outcome. Our results indicate that PRs and implantation rates are poorer in women with higher endometrial echogenicity in comparison with those with lower endometrial echogenicity. Several characteristics differentiate the present study from others previously published on the same topic (1 20). Participants were controlled for age ( 8 years), the absence of 278 Fanchin et al. Endometrial echogenicity and receptivity Vol. 74, No. 2, August 2000

6 uterine abnormalities, and the number and quality of embryos transferred ( 2). Women whose uterine position impaired adequate visualization of endometrial texture were excluded. The assessment of endometrial echogenicity was performed objectively with a computer-assisted image analysis system. This allowed us to avoid the use of arbitrary and subjective classifications and to sort data accumulated from 228 IVF- ET cycles into multiple groups according to the precise degree of upward extension of hyperechogenic transformation. The strict methodology and the similarity of all echogenicity groups with regard to population, COH, and embryology characteristics allow us to conclude that there is a relation between the degree of endometrial hyperechogenicity on the day of hcg and PRs. Hence, the present results confirm and extend earlier observations that the echogenic status of the endometrium assessed in the late follicular phase predicts endometrial receptivity status (9, 10, 1 18). The histologic bases for the upward extension of endometrial echogenicity remain, however, unknown. Previous reports have shown that echogenicity status reflects the degree of histologic development of the endometrium (6 8, 11). Therefore, the degree of extension of hyperechogenic transformation of the endometrium observed on the day of hcg administration may denote an acceleration of secretory changes and a proportional forward slide of the period of endometrial receptivity. In the follicular phase of the menstrual cycle, hyperechogenic endometrial features are infrequent (21). During this phase, endometrium-to-myometrium interfaces and the uterine cavity appear hyperechogenic, whereas the remaining part of the endometrial thickness displays a hypoechogenic or isoechogenic signal in relation to the surrounding myometrium (6 11). This US aspect may be a reflection of the glandular straightness, reduced glandular secretion, and/or reduced stromal edema that characterize the proliferative endometrium, with a decreased number of interfaces on US. During the luteal phase, a progressive base-to-surface extension of endometrial hyperechogenicity is seen in natural cycles (6 8), in E 2 P replacement cycles (11), and in COH cycles (9, 10). This probably is a result of the endometrial exposure to P, although it is conceivable that additional endocrine or paracrine autocrine mechanisms participate in this process. We recently reported that premature exposure of the endometrium to P during the follicular phase leads to a faster progression of endometrial echogenicity during the early luteal phase in COH cycles (12). These data further support the effects of P on endometrial echogenicity. The histologic bases for the increase in endometrial echogenicity are still debated. Stromal edema is thought to be the key event responsible for hyperechogenic signals (11). Indeed, the contact of interstitial fluid with glands and vessel walls is likely to generate echoes in the endometrium and, therefore, increase endometrial echogenicity (11). However, some arguments challenge this hypothesis. During the menstrual cycle, stromal edema occurs approximately on days (22). It is therefore a relatively late feature in the timed cascade of the histologic transformation of the endometrium. Even the unbalanced hormonal milieu induced by COH and its putative consequences on the endometrium are probably insufficient to boost endometrial secretory transformation to such a degree. Indeed, the present data indicate a remarkable advancement of hyperechogenic transformation in a large fraction of women, often 50% of the endometrium on the day of hcg administration. Therefore, other histologic events, such as glandular coiling (day 16 onward) and/or secretion (day 17 onward), may contribute to the increase in endometrial echogenicity. As a result of endometrial exposure to supraphysiologic P levels during the follicular phase of COH (5, 12), these early postovulatory histologic features theoretically may occur prematurely on the day of hcg administration. However, the lack of correlation between circulating P levels and endometrial echogenicity values, observed in previous investigations (15, 21) and confirmed by the present study, is inconsistent with this hypothesis. Direct effects on the endometrium of other hormones, such as androgens (2) or exogenous gonadotropins (), constitute alternative mechanisms to explain the premature increase in endometrial echogenicity during the late follicular phase of COH. This latter possibility, however, requires further examination. The present study showed a progressive decrease in PRs and implantation rates with the extension of endometrial echogenicity on the day of hcg. The perception of this gradual phenomenon was possible because of the optimal sensitivity and precision of our measurements. The strength of the correlation between endometrial texture patterns, histologic findings, and IVF-ET outcome is susceptible to variation according to subjective evaluation of scans (24) and the use of arbitrary classifications (10, 1, 14, 18). The computer-assisted image analysis module (IôDP) used in this study was developed specifically for assessment of the degree of hyperechogenic transformation of the endometrium and improved both the reliability and objectivity of our calculations. Another computer-assisted analysis of the endometrium was reported by Leibovitz et al. (25). Their system was based on the calculation of the intensity of echogenic signals relative to the surrounding myometrium. These investigators reported a progressive and significant increase of endometrium-to-myometrium relative echogenicity coefficients during the luteal phase of spontaneous and COH cycles (26). The progressive decrease in PRs observed in the present FERTILITY & STERILITY 279

7 study leads us to speculate that the greater the submyometrial hyperechogenicity, the greater the histologic transformation of the endometrium and, probably, the lower the endometrial receptivity. Our observation that PRs remained 10% even in the 70% echogenicity group suggests that other factors, possibly linked to embryo quality, may compensate for the reduction in endometrial receptivity (5) and allow ongoing pregnancies. Additional investigation of unselected patients with poor-quality embryos will clarify the possibility of a more drastic decrease in implantation rates in hyperechogenic groups. It is also noteworthy that endometrial thickness was not correlated with plasma E 2 levels on the day of hcg administration in the present analysis. Indeed, time-dependent (rather than concentration-dependent) (21) thickening of the endometrium occurs throughout the follicular phase of the menstrual cycle (21, 26), probably because of the proliferative action of estrogens. The present data confirm the hypothesis that supraphysiologic E 2 levels induced by COH do not accelerate either the pace or the magnitude of endometrial development (21) beyond values triggered by physiologic concentrations of estrogens. Further, tissue and/or vascular mechanisms may concur to modulate the action of estrogens on the endometrium. Indeed, the present and previously published series (6, 9, 10, 12, 1, 16, 17 21, 26) showed conspicuous intersubject variability in endometrial thickness during the late follicular phase. Incidentally, the present study failed to identify any relation between endometrial thickness on the day of hcg administration and IVF-ET outcome. In conclusion, the present data indicate that the more advanced is the hyperechogenic transformation of the endometrium at the time of hcg administration, the lower are the PRs and implantation rates in IVF-ET. The mechanism put forth to explain this inverse relation is an alteration of the endometrial receptivity, which presumably results from hastened secretory transformation of the endometrium. It is possible that the low PRs observed in the hyperechogenic groups in this selected series may decrease even further in older women with poor-quality embryos. In these cases, canceling the cycle or delaying ET by cryopreserving embryos may be a judicious alternative. Antiprogestin administration (27) in the late follicular phase of COH to reduce the possible P-induced acceleration of endometrial hyperechogenicity may be an attractive measure to restore endometrial receptivity. However, these issues deserve further investigation. Acknowledgment: The authors thank Organon Pharmaceuticals, Saint Denis, France, for making possible the use of sophisticated ultrasonography equipment in the present work. References 1. Navot D, Laufer N, Kopolovic J, Rabinowitz R, Birkenfeld A, Lewin A, et al. 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