Endometrial thickness and growth during ovarian stimulation: a possible predictor of implantation in in vitro fertilization*

Vol. 52, No.3, September 1989

FERTILITY AND STERILITY

Printed on acid.free paper in U.S.A.

Copyright © 1989 The American Fertility Society

Endometrial thickness and growth during ovarian stimulation: a possible predictor of implantation in in vitro fertilization*

Yael Gonen, M.D.t Robert F. Casper, M.D.:\: William Jacobson, Ph.D. Joseph Blankier, M.D. Division of Reproductive Science, University of Toronto, Toronto, Ontario, Canada

The present study was undertaken to evaluate endometrial thickness and the amount of endometrial growth (~) in patients who conceived during in vitro fertilization (IVF) (n = 36) compared with matched women who did not conceive (n = 72). Estradiol (E 2) and endometrial thickness were measured daily from cycle day 10 to the day after human chorionic gonadotropin (hCG). Mean endometrial thickness and E2 levels on cycle day 10 did not differ. On the day before ovum retrieval, significantly thicker endometrium was observed in the pregnant than in the nonpregnant women (8.6 ± 0.3 [SEM] and 7.1 ± 0.3 mm, respectively; P < 0.0005), whereas the mean E2 levels did not differ. The ~ endometrial growth was greater in the women who conceived than in the nonpregnant group (4.3 ± 0.2 and 2.5 ± 0.2 mm, respectively; P < 0.0005). The fertilization rate and serum E2 levels did not correlate with endometrial thickness nor with ~ endometrial growth. Our data suggest that the amount of endometrial growth during ovarian hyperstimulation and the endometrial thickness on the day before oocyte retrieval deserve further study as possible predictive parameters for implantation. Fertil Steril 52:446, 1989

Vaginal sonography has significantly influenced fertility management and greatly extended the role of ultrasound in gynecology. The transvaginal transducer enables very detailed visualization of the uterine cavity. Changes in the thickness and texture of the endometrium have been observed during follicular maturation in spontaneous 1 and stimulated cycles. 2- 4 Different patterns of endometrial response have been described,1,3 but the precise relationship between endometrial thickening

and reflectivity to ultrasound has not yet been clarified. This study was undertaken to evaluate endometrial thickness and the amount of endometrial growth or thickening (Il) in women who conceived in our in vitro fertilization (IVF) program, compared with matched women who failed to conceive, to discern whether there were certain endometrial parameters that were associated with the subsequent occurrence of pregnancy.

Received February 9, 1989; revised and accepted April 25, 1989. * Supported by a Medical Research Council of Canada grant to Robert F. Casper and by a Wyeth Ltd. Fellowship to Yael Gonen, M.D. t Visiting clinical research fellow from the Department of Obstetrics and Gynecology, Carmel Hospital, Haifa, Israel. ':j: Reprint requests: Robert F. Casper, M.D., 6-240 EN, Toronto General Hospital, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4.

MATERIALS AND METHODS

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From January 1988 to December 1988 inclusive, IVF cycles in 36 women resulted in a clinical pregnancy. A control group of 72 nonpregnant women was selected by choosing 2 women who did not conceive during an IVF cycle in the same week as each conception cycle, and as close to the same day as possible. Fertility and Sterility

ness. We also determined the number of oocytes retrieved, the fertilization rate, and the number of embryos transferred. The correlation between e n - . dometrial thickness and the fertilization rate in each patient was also determined. Estradiol Assay

Figure 1 Ultrasound image of the measurement of endometrial thickness using a 7-MHz transvaginal transducer. The maximum thickness of the endometrium on both sides of the midline was measured by determining the distance between the two markers as illustrated in the plane through the central longitudinal axis of the uterine body.

Ovarian follicular stimulation was routinely induced with clomiphene citrate [CC (Serophene; Serono, Randolph, MA)], 100 mgjday, cycle days 5 to 9, and human menopausal gonadotropin (Pergonal; Serono), 75 to 150 IV intramuscularly (1M) daily starting on cycle day 5 or 6. Human chorionic gonadotropin [hCG (Profasi; Serono)], 5,000 IV 1M, was routinely used to trigger the final stage of follicular maturation. The response to treatment was monitored daily from day 10 of the menstrual cycle by transvaginal ultrasound measurements of follicular diameters and endometrial thickness, together with daily determination of serum levels of estradiol (E 2) and luteinizing hormone (LH) by radioimmunoassay (RIA). Endometrial thickness was measured on the "frozen" ultrasound image with a 7-MHz transvaginal transducer (Type 8538; Bruel & Kjaer, Naerum, Denmark). The maximum thickness of endometrium on both sides ofthe midline was measured in the plane through the central longitudinal axis of the uterine body (Fig. 1). Oocyte retrieval was performed approximately 34 hours after hCG administration by transvaginal ultrasound-guided follicular aspiration. IVF and embryo transfer (ET) were performed using standard techniques.5 The pregnancy and control groups were compared by determining mean serum E2 and endometrial thickness on cycle day 10 and on the day after hCG administration. We determined the amount of growth 'of the endometrium anp the change in E2 levels from day 10 until the day after hCG administration, and we calculated the correlation ofE2 levels with endometrial thickVol. 52, No.3, September 1989

Estradiol was measured by a solid-phase radioimmunoassay (RIA) using 1251-labeled estradiol (Coat-A-Count Estradiol; Diagnostic Products Corporation, Los Angeles, CA). E2 values were expressed in pmol/L (pgjmL X 0.003671). Intra-assay and interassay coefficients of variations were also determined directly in serum pools placed at two or three levels of the working range, respectively. The intra-assay coefficient of variation at 290 and 1,000 pmoljL of E2 was 5.6% and 4.0%, respectively. The interassay coefficient of variation at 280, 500, and 2,500 pmol/L was 12%, 9.5%, and 7.5%, respectively. Statistical Evaluation

Hormone concentration, endometrial thickness, number of oocytes retrieved, and embryos transferred were compared by Student's t-test for unpaired results. Fertilization rates were analyzed using the nonparametric Mann-Whitney V-test on ranked data. Correlation between hormone concentration and endometrial thickness was determined by linear regression analysis, whereas correlation between fertilization rates and endometrial thickness was evaluated by Spearman rank correlation coefficient test. P values less than 0.05 were regarded as statistically significant. Data are presented throughout this paper as a mean ± standard error of the mean (SEM). RESULTS

Age, gravidity, parity, cause of infertility, and protocol used for stimulation were similar in the 36 pregnant and 72 nonpregnant women. Table 1 presents the mean endometrial thickness and serum E 2 levels on cycle day 10 and on the day after hCG administration. Neither endometrial thickness nor serum E 2 levels were different between the two groups on cycle day 10. In contrast, on the day after hCG administration, endometrial thickness was significantly greater in the pregnant women than in the nonpregnant women [8.6 ± 0.3 and 7.1 ± 0.3 mm, respectively; P < 0.0005], whereas no differences were observed in serum E 2 levels (5,776 Gonen et aI.

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Table 1 Endometrial Thickness and E2 Concentrations in IVF Patients Who Conceived and in Matched Controls Who Did Not Conceive

Pregnant (n = 36) Control (n = 72)

E 2-day 10

Endometrial thickness afterhCG

E 2 day afterhCG

mm

pmol/L

mm

pmol/L

mm

pmol/L

4.3 ± 0.2

2,164 ± 177

8.6 ± 0.3 a

5,776 ± 399

4.3 ± 0.2a

3,612 ± 370

4.6 ± 0.2

2,145 ± 139

7.1 ± 0.3

5,836 ± 359

2.5 ± 0.2

3,647 ± 285

Endometrial thickness day 10

~

Endometrial thickness

~E2levels

a P<0.0005.

± 399 and 5,836 ± 359 pmol/L in the pregnant and nonpregnant women, respectively). The mean ~ endometrial thickness from cycle day 10 to the day after hCG was significantly greater in the pregnant women than in the nonpregnant women (4.3 ± 0.2 and 2.5 ± 0.2 mm, respectively; P < 0.0005), whereas ~E2 levels did not differ and were 3,612 ± 370 and 3,647 ± 285 pmoljL, respectively. No correlation between serum E2 levels and endometrial thickness was found on cycle day 10 or on the day after administration of hCG in either group of patients. No differences were observed in the mean number of oocytes retrieved per patient in the pregnant and nonpregnant women (4.6 ± 0.3 and 5.2 ± 0.4, respectively; Table 2). The fertilization rate in the pregnant women was significantly higher (80.8% ± 3.4% versus 57.2% ± 3.3%; P < 0.0005), resulting in significantly more embryos transferred per patient (3.4 ± 0.2 and 2.7 ± 0.2 in the pregnant and nonpregnant women, respectively; P < 0.025). No correlation was found between endometrial thickness or ~ endometrial thickness and fertilization

Table 2 Mean Number of Oocytes Retrieved, Embryos Transferred, and Fertilization Rate in IVF Patients Who Conceived or in Matched Controls Who Did Not Conceive No. of ova retrieved

Fertilization rate

No. of embryos transferred

%

Pregnant (n = 36) Control (n = 72)

.

4.6±0.3

80.8 ± 3A a

304 ± 0.2b

5.2 ± 004

57.2 ± 3.3

2.7 ± 0.2

a P<0.0005. b P < 0.025.

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rate in the pregnant group (p = 0.035 and 0.05, respectively) or in the nonpregnant group (p = -0.06 and -0.06, respectively). When data for the whole group of 108 patients (pregnant and nonpregnant combined) were examined, no correlation was seen between fertilization rate and endometrial thickness or ~ thickness (p = 0.103 and 0.2, respectively).

DISCUSSION

The introduction of ultrasound transducers suitable for transvaginal applications, particularly in their higher-frequency versions, represents a step forward in gynecological sonography. The present study reports vaginal ultrasound measurements of endometrial thickness and endometrial growth in pregnant and nonpregnant patients undergoing ovarian stimulation for IVF. We demonstrate by means of this sensitive technique that endometrial thickness and ~ endometrial thickness are increased significantly in pregnant versus nonpregnant cycles. These results differ from the results reported so far using transabdominal sonography,3,6-9 in which only one report9 demonstrated a small but significant increase in endometrial thickness in a small group of pregnant patients compared with nonpregnant patients. All other investigators3,6-8 failed to find a significant difference in the mean endometrial thickness of conception versus nonconception cycles. We consider the transvaginal approach with a high-frequency transducer to have several advantages over the transabdominal route. Because of the proximity of the vaginal transducer to the uterus, the high-resolution pictures obtained enable very detailed visualization of the uterine cavity and accurate measurement of small changes in Fertility and Sterility

endometrial thickness during the cycle. Because the examination is done with an empty bladder, there is no concern that a distended bladder may distort the shape of the uterine cavity. Our data show that the endometrial growth during ovarian hyperstimulation and the endometrial thickness on the day before ovum retrieval are significantly greater in conception than in nonconception cycles and suggest that endometrial changes during IVF stimulation may eventually be useful as predictive parameters for implantation. The results of our study contrast with observations by Welker et a1. 10 and Fleischer et alY who demonstrated, using transvaginal ultrasound, that the thickness of the endometrium did not correlate with the occurrence of pregnancy. The reason for the difference between these studies and ours is not clear, but may be related to either the smaller number of patients in the previous reports or difference in measurement technique. In our study, endometrial thickness, ~ endometrial thickness, and fertilization rate were all significantly elevated in pregnant patients compared with those who did not conceive. It is possible that both endometrial growth and fertilization rate may have predictive value regarding implantation and subsequent pregnancy. However, endometrial thickness is measured on the day after hCG administration (or followed from day 10 to the day after hCG for ~ thickness), and thus if definite confidence limits can be established, will allow cancellation of unfavorable cycles before oocyte recovery by either ultrasound or laparoscopy. The fertilization rate is not helpful in the same fashion, because it can only be calculated after the patient has already been subjected to a surgical procedure for oocyte recovery. The reason for increased endometrial thickness in the pregnant cycles compared with the nonpregnant cycles is not clear. However, the observation that fertilization rate and endometrial thickness are not correlated indicates that these two measurements are independent of each other. One factor that may influence fertilization rate is the degree of follicular and oocyte maturity. It is possible that endometrial thickness is, therefore, independent of the degree of maturation of the follicles, a suggestion that is also supported by the absence of correlation between endometrial thickness and serum estrogen levels. .A possible mediator of endometrial growth is the level of estrogen receptor activity in the endometrium itself. It is possible that the administration Vol. 52, No.3, September 1989

of CC, which has been shown to deplete estrogen receptors,12-14 may influence both endometrial growth and implantation. 15 In some patients, endometrial estrogen receptors may be depleted to a greater extent than others, resulting in less endometrial growth in relation to serum estrogen levels and, subsequently, a thinner endometrium on the day after hCG administration. This suggestion is supported by studies in IVF patients demonstrating a high incidence of histological and ultrastructural endometrial abnormalities in CC-stimulated cycles compared with natural cycles or cycles stimulated with hMG and hCG alone. 16 In contrast, Markiewicz et alP demonstrated that CC, under in vitro conditions, exerts a mainly estrogenic effect on the endometrium, whereas others 18.19 reported no specific deleterious histological or ultrastructural changes in the endometrium after CC administration. The endometrial response in patients undergoing ovarian hyperstimulation can be seen on sonographic examination as a quantitative change in thickness and also as a qualitative change in appearance or reflectivity. Different endometrial patterns on sonography have been described according to hyper- and hypoechogenic layers 10 and general ultrasound reflectivity compared with the myometrium. 3 The precise relationship between endometrial thickness and reflectivity has not yet been clarified, and we did not attempt to evaluate endometrial texture in the present study. In general, no agreement exists between different authors as to which endometrial pattern correlates best with pregnancy, and we believe that both endometrial thickness and ultrasound reflectivity deserve further investigation as possible parameters for an intrauterine environment favorable for implantation.

Acknowledgment. We thank Ms. Mary Coates for her help with the manuscript.

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