Predicting factors for endometrial thickness during treatment with assisted reproductive technology

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1 Predicting factors for endometrial thickness during treatment with assisted reproductive technology Wiser Amir, M.D., a Baum Micha, M.D., a Hourwitz Ariel, M.D., a Lerner-Geva Liat, M.D., Ph.D., b Dor Jehoshua, M.D., a and Shulman Adrian, M.D. a a IVF Unit, Department of Obstetrics and Gynecology, and b Women and Children s Health Research Unit, Gertner Institute for Epidemiology and Health Policy Research, Chaim Sheba Medical Center, Tel Hashomer, Israel Objective: To discover the factors contributing to endometrial thickness, and to assess the impact of endometrial thickness on pregnancy rates (PRs) according to these factors. Design: Retrospective study. Setting: In vitro fertilization unit in a university hospital. Patient(s): All women with primary infertility and no previous pregnancies who underwent IVF treatment at the Chaim Sheba Medical Center, Tel Hashomer, Israel, between August 9, 2001 December 31, Intervention: Measurement of endometrial thickness by the use of transvaginal ultrasound probe on the day that hcg was administered during an IVF cycle. Main Outcome Measure(s): Factors influencing endometrial thickness and the relationship between endometrial thickness and PRs. Result(s): The mean endometrial thickness decreased as a function of the patient s age. The thickest endometrium was found in patients 25 years of age ( mm), and the thinnest endometrium was found in patients 40 years of age ( mm). Other factors, such as E 2 levels, etiology of infertility, induction of ovulation protocol, and type of gonadotropin used, were also found to contribute to endometrial thickness. Conclusion(s): Our data support the case for an aging of the endometrium. The chances of achieving a thick endometrium for patients 40 years of age are lower than for younger patients. Furthermore, a thicker endometrium is correlated with a higher PR only for patients 35 years of age. (Fertil Steril 2007;87: by American Society for Reproductive Medicine.) Key Words: Endometrial thickness, IVF, ICSI, pregnancy, age In recent years, many studies have evaluated the effect of endometrial thickness and pattern on pregnancy rates (PRs) in assisted reproductive technology (ART) patients (1 10). The results obtained were controversial. Some authors demonstrated a higher probability of pregnancy once the endometrium attained a threshold thickness (2, 3, 9 11), while others failed to recognize a significant correlation between endometrial thickness and PRs in ART patients (4, 6, 7). Other authors referred to discriminatory zones of 7 mm with no pregnancies (1), and of 14 mm with a significant reduction in implantation rate and PR (5). Except for intrauterine adhesions and E 2 levels, no factors were found that could be implicated in the variability of endometrial thickness during ART cycles. The primary aim of the present study is to assess the factors contributing to endometrial Received November 21, 2005; revised September 2, 2006; accepted November 3, The present address of Shulman Adrian, M.D., is the IVF Unit, Department of Obstetrics and Gynecology, Meir Medical Center, Kfar Saba 44281, Israel. The Chaim Sheba Medical Center, Tel Hashomer, Israel, is affiliated with the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Reprint requests: Wiser Amir, M.D., IVF Unit, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel (FAX: ; wiserniv@netvision.net.il). thickness. The secondary aim is to evaluate the impact of endometrial thickness on PRs. MATERIALS AND METHODS We reviewed the medical records of all IVF and intracytoplasmic sperm injection (ICSI) cycles performed in our IVF unit from August 9, 2001 to December 31, All data were obtained from our computerized database. Information was entered online (real time) during the various stages of each treatment cycle. The study was approval by the Ethics Committee of our hospital. To avoid the impact of previous abortions, only women with no previous pregnancies were included. We included only stimulated cycles that achieved ovum pickup (OPU) and ET. Nonendometrial parameters analyzed were age of patients, etiology of infertility, protocol of treatment, medication for induction of ovulation, peak levels of E 2, and number of retrieved eggs. During this period, we used three protocols for induction of ovulation in our IVF patients: the long protocol, the short protocol, and the GnRh antagonist protocol. The protocol for each patient was chosen by the treating physician based on clinical judgment and, when possible, in accordance with the patient s performance in previous cycles. The medications for induction of ovulation were either recombinant FSH (Gonal-F Serono, Aubans, The Netherlands or Puregon, NV /07/$32.00 Fertility and Sterility Vol. 87, No. 4, April 2007 doi: /j.fertnstert Copyright 2007 American Society for Reproductive Medicine, Published by Elsevier Inc. 799

2 Organon, Oss, The Netherlands), or purified urinary FSH LH (Menogon Ferring SA, Saint-Prex, Switzerland). Endometrial thickness was measured by transvaginal ultrasound probe on the day of administration of hcg. Measurements were conducted in the midsagittal plane, from the outer edge of the endometrial-myometrial interface to the outer edge of the widest part of the endometrium. The pregnancy test was performed 12 days after ET. Pregnancy was defined according to the hcg level required, i.e., at least three times higher than the baseline level ( 30 IU/mL). Data were analyzed using SAS software (SAS Institute Inc., Cary, NC). Comparisons of proportions were made using the chi-square test. The linear trend was tested by the Mantel-Haenszel test. Continuous variables are presented as means and SD, and were tested by Student s t-test or analysis of variance. All tests were two-tailed, and P 0.05 was considered statistically significant. To determine correlations between the development of an endometrium 7 mm, patients characteristics, and treatment parameters, a stepwise logistic regression analysis (including age, etiology of infertility, treatment protocol, medication, and level of E 2 ) was performed. The number of retrieved eggs was not included in the model because of the strong association with E 2 level. RESULTS During the study period, we performed 4,518 (fresh) cycles of OPU-ET. We excluded from the present analysis 385 spontaneous cycles, 1,285 cycles from women with past pregnancies, 87 natural protocol cycles, 52 cycles in cancer patients, and 370 cycles in which no endometrial measurements were performed, leaving 2,339 cycles for analysis. The mean age of patients was years (range, years), and 35% of patients were 35 years of age. The mean endometrial thickness was mm (range, mm). Endometrial thickness in the majority of patients (86.9%) was 7 14 mm. In 163 patients (7.0%), endometrial thickness was 7 mm. Patients who became pregnant had a thicker endometrium ( mm) compared to patients who did not become pregnant ( mm) (P.006). Table 1 summarizes PRs according to endometrial thickness and patients age. A thicker endometrium contributed to higher PRs only in patients 35 years of age. We found that the younger the patient, the thicker the endometrium (Table 2). The thickest endometrium was found in patients 26 years of age ( mm) (P). The thinnest endometrium was found in patients 40 years of age ( mm). The etiology of infertility influenced endometrial thickness: women with a male-factor etiology of infertility had a thicker endometrium compared to all other causes of female infertility ( mm and mm, respectively; P). The short protocol of induction of ovulation was associated with the thinnest endometria ( mm), followed by the antagonist protocol ( mm). The long protocol of induction of ovulation yielded the widest endometria ( mm; P). Recombinant FSH or recombinant FSH combined with urinary gonadotropins resulted in thicker endometria ( mm) compared to urinary gonadotropins alone ( mm; P). These effects were independent of age. When we compared the impact of the protocols containing HMG and antagonist versus protocol recombinant FSH and the long protocol on the endometrial thickness of patients above and below age 40 years, the former yielded thinner endometria in both groups. We found that E 2 levels were correlated with endometrial thickness only in patients with low estrogen levels ( 1,000 pg/ml). For this group of patients, endometrial thickness was mm, significantly thinner than in all other groups of patients (P). As shown in Table 2, a positive correlation was found between the number of retrieved eggs and endometrial thickness, with the thinnest endometria in cases in which only one egg was aspirated ( mm), and the thickest endometria ( mm) in cases in which 6 eggs were retrieved (P). A stepwise logistic regression analysis (Table 3) for variables correlated with an endometrial thickness of 7 mm revealed TABLE 1 Pregnancy rate according to age and endometrial thickness. 1 7 mm (n 163) mm (n 2,032) mm (n 144) n % n % n % P value (for trend) Age (y) a 35 (n 1,518) 18/ /1, / (n 816) 10/ / / Total (n 2,334) 28/ /2, / a Missing ages, n 5. Amir. Endometrial thickness predictors during ART. Fertil Steril Amir et al. Endometrial thickness predictors during ART Vol. 87, No. 4, April 2007

3 TABLE 2 Parameters influencing endometrial thickness. Characteristics N Endometrial thickness 7 mm mm >14 mm (n 163) (%) a (n 2,032) (%) a (n 144) (%) a Mean endometrial thickness (mm) P value b All cycles 2, Age (y) , Missing 5 Etiology of infertility Male only Female 1, Protocol Long 1, Short Antagonist Missing 43 Medication FSH HMG FSH HMG 1, E 2 level (pg/ml) 0 1, ,001 1, ,501 2, , Missing 17 No. of retrieved eggs , Missing 100 a Percentage of 7, , and 14 in each subgroup. b P for mean endometrial thickness and for percentage in each characteristic. Amir. Endometrial thickness predictors during ART. Fertil Steril that patients 40 years of age had an odds ratio (OR) of 5.58 for developing a thin endometrium, compared to patients 26 years of age. A female factor as the main etiology of infertility had an OR of 2.71 for a thin endometrium, compared to the male factor only. Treatment protocol and medication for induction of ovulation were also associated with an endometrial thickness 7 mm. The level of E 2 did not contribute significantly to the development of a thin endometrium (Table 3). DISCUSSION The association between endometrial thickness and cycle outcome was evaluated for several groups. In our population, the 5th percentile of endometrial thickness was between 6 7 mm, and the 95th percentile was between mm. Because it is impractical to measure intervals 1 mm, we chose the cutoffs of 7 mm (7%) and 14 mm (94%) (within the limits of 2 SD). Yoeli et al. (12) also defined the same lower limit of appropriate endometrial thickness for patients in ART programs. In their study, the two margins also constituted the 5th and 95th percentile values. These differentiations of the endometrial thickness are correlated with the data describing low PRs for endometrial thickness 7mm(1), and higher PRs for thickness 14 mm (13). Unlike Weissman et al. (5), we did not find an adverse outcome for endometrial thickness 14 mm. Fertility and Sterility 801

4 TABLE 3 Logistic regression for developing endometrium of <7 mm. OR 95% confidence interval P value Age (y) Etiology Male only 1.00 Female Protocol Long 1.00 Short Antagonist Medication FSH, or FSH HMG 1.00 HMG E 2 level (pg/ml) 0 1, ,001 1, ,501 2, , Amir. Endometrial thickness predictors during ART. Fertil Steril We found that a thicker endometrium correlated with higher PRs (Table 1) in patients 35 years of age. This correlation between endometrial thickness and PRs was not observed among patients 35 years of age. Maternal age is a well-known determinant of fertility and treatment outcome in IVF patients (14 16). The decrease in fertility with age is attributed mainly to the quality of oocytes, and we postulate an additional impact of age on uterine receptiveness as a cofactor for decreasing fertility in older patients. Early rise in progesterone level during the IVF cycle has a negative impact on the endometrium. These rise do not affect the quality of eggs, as revealed by Shulman et al. (17); PRs decreased in egg donors with an elevation of plasma levels of P before administration of hcg. The PRs of recipients from the same cycle were not affected. All recipients were menopausal, and received estrogens only during the follicular phase. There was no elevation of P in any of the recipients before the administration of exogenous P. This observation can explain the relative high PR among older patients on oocyte donation programs. Their endometrium is not adversely affected by the rise in levels of P. Our study is unique in performing a stepwise regression analysis to find prognostic factors for a thin endometrium, and in attempting to consider all aspects of treatment and their influence on the endometrium. Our results emphasize an aging of the endometrium, independent of ovarian aging (expressed by maximum levels of E 2 and number of retrieved eggs). Thus the chances of achieving a thick endometrium in patients 40 years of age are lower than for younger patients. Our results contradict the data published by Zhang et al. (18) showing that endometrial thickness is dependent on serum E 2 level, and independent of patient s age. A possible explanation for the differences between these studies is the relatively small sample of data in Zhang et al. (18). Gurbuz et al. (19) studied factors influencing endometrial thickness in postmenopausal women (aged years). They also described a negative association between endometrial thickness and age (19). Our data imply that the aging of the endometrium starts sooner, during the fertility period. A possible explanation for the aging of the endometrium was suggested by Gruninger et al. (20) and Hsueh et al. (21). In their animal model, endometrial blood vessels were affected by angiosclerotic changes, characterized by mild to moderate perivascular and intimal sclerosis. The incidence and severity of angiosis increased with advancing age, in parallel with stromal angiosclerosis (20). A general decline in uterine estrogen receptors was found in female rats with advancing age (21). Endometrial glandular and stromal apoptosis was found to increase with age (22). These age-related changes could serve as a partial explanation for the decrease in endometrial thickness with the age. The connection between level of serum E 2 and endometrial thickness is well-known (18, 23). As found in other 802 Amir et al. Endometrial thickness predictors during ART Vol. 87, No. 4, April 2007

5 studies, we also found that E 2 levels have a positive effect on endometrial thickness. The new element in our study is the discovery of a threshold level, around 1,000 ng/ml, differentiating between a thin endometrium and a normal-range endometrium. According to our data, higher E 2 levels, i.e., 1,000 ng/ml, have no further significant impact on endometrial thickness. Our finding of appropriate endometrial thickness in patients with male factor as the single etiology of infertility points to a possible relationship between the endometrial profile and any other female-related etiology of infertility. Other factors influencing the endometrium are the treatment protocol of induction of ovulation and the specific medication administered for this purpose. Human menopausal gonadotropin yielded a thinner endometrium (10.1 mm) compared to recombinant FSH (10.7 mm). The explanation for this observation could be that the LH contained in the HMG enhances androgen production. The subtle elevation in androgen production may impair the proliferation of endometrium. Treatment of menopausal woman with a combination of estrogen and androgen induces severe stromal compaction and glandular atrophy in the endometrium (23). Testosterone and danazol directly inhibit human endometrial-cell proliferation in tissue culture (24, 25). Elevation of androgen may specifically antagonize estrogen action directly in the endometrium (26). There is evidence to suggest that PRs in an antagonist treatment cycle may be somewhat lower than in a cycle using agonists in the long protocol (26, 27), possibly because GnRH antagonists may influence the mitotic programming of cells included in endometrial development (28). These effects of HMG and antagonist protocol on the endometrium were observed in younger patients, as well as in patients 40 years of age. We doubt whether a difference of 0.7 mm has any clinical meaning in the majority of patients, though it may be important for patients with repeated endometrial problems. In this group of patients, avoiding the use of protocols with antagonists and HMG may improve the thickness of the endometrium. The results of our analysis require further evaluation, and may eventually lead to a change in the policy of induction of ovulation for older patients. In conclusion, we found an aging of endometrium with a negative effect of these thin endometrium only in patients 35 years of age. Acknowledgment: We thank Ms. Boyko Valla for indispensable help with statistics. REFERENCES 1. Isaacs JD Jr, Wells CS, Williams DB, Odem RR, Gast MJ, Strickler RC. Endometrial thickness is a valid monitoring parameter in cycles of ovulation induction with menotropins alone. Fertil Steril 1996;65: Noyes N, Liu HC, Sultan K, Schattman G, Rosenwaks Z. 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