Effect of estradiol valerate on endometrium thickness
during clomiphene citrate-stimulated ovulation
Chonthicha Satirapod1...
overall intrauterine health.14
Endometrial thickness
was also shown to positively correlate with receptive-
ness. Previous...
Transvaginal sonographic examination
After aligning the uterus along with the central longi-
tudinal axis, the endometrial...
the maximum follicular diameters were comparable in
both placebo and EV added groups in CC-stimulated
cycles (Table 3).
effects, or produce new adverse effects that were not
observed in CC stimulation alone.
Therefore, adding the oral form of...
21. El-Toukhy T, Taylor A, Khalaf Y et al. Pituitary suppression in
ultrasound-monitored frozen embryo replacement cycles....
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Jog12130 effect of estradiol valerate on endometrium thickness


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Jog12130 effect of estradiol valerate on endometrium thickness

  1. 1. Effect of estradiol valerate on endometrium thickness during clomiphene citrate-stimulated ovulation Chonthicha Satirapod1 , Siripen Wingprawat1 , Rattiya Jultanmas2 , Sasivimol Rattanasiri3 , Siwanon Jirawatnotai4 and Wicharn Choktanasiri1 Departments of 1 Obstetrics and Gynaecology and 2 Nursing, 3 Research Center, Faculty of Medicine, Ramathibodi Hospital, and 4 Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand Abstract Aim: The aim of this study was to examine the effects of estradiol valerate (EV) on the thickness of clomiphene citrate (CC)-stimulated endometrium. Material and Methods: Thirty-four normal ovulatory women were randomized double-blindly into two groups to receive CC 100 mg/day on day 2–6 of the treatment cycle, and either vitamin B (placebo) or EV 6 mg/day on day 10–14 of the cycle. The endometrial thickness, endometrial pattern, numbers of mature follicles, and maximal diameters of preovulatory follicles were evaluated by transvaginal sonographic examination. Results: Thirty women completed both treatment cycles. Two other participants dropped out during the treatment due to side-effects (headache). The average endometrial thickness of the group treated with CC + placebo became slightly thinner when compared to the thickness at the baseline (9.04 vs 9.52 mm; P = 0.24). The CC + placebo and the CC + EV resulted in similar endometrial pattern, ovulation day, numbers of mature follicles, and sizes of the leading follicles before ovulation. However, an addition of EV into the CC cycle significantly increased the average endometrial thickness (10.7 mm vs 9.04 mm; P < 0.001). Conclusions: We concluded that the addition of 6 mg/day EV following the CC treatment can prevent the endometrial thinning without perturbing folliculogenesis and ovulation. Key words: clomiphene citrate, endometrial thickness, estradiol valerate, folliculogenesis, ovarian stimulation. Introduction Clomiphene citrate (CC) is an orally active non- steroidal estrogen agonist/antagonist that is widely used as the first-line ovulation inducer in anovulatory women.1,2 Nowadays, CC is useful in a variety of indi- cations, such as induction of superovulation in unex- plained infertility3 and the stimulation protocol for in vitro fertilization (IVF) cycles.4–6 CC has several marked advantages for these indications, because it is highly effective in inducing ovulation, while relatively safe, inexpensive and can be administered orally.1 However, despite the fact that CC administration in anovulatory patients can improve ovulation rate by 60–85%, the pregnancy rate linked to CC treatment is noticeably lower than expected (only around 30–40%).4 This dis- crepancy is believed to result from CC’s negative effect on the cervical mucus quality7,8 and its anti-estrogenic activity.9–11 In fact, it has been reported that the anti- estrogenic activity of CC can cause thinning of endometrium.9,10,12,13 Normally, endometrial thickness is a reflection of its ability to proliferate and of the Received: November 8 2012. Accepted: March 14 2013. Reprint request to: Dr Chonthicha Satirapod, Department of Obstetrics and Gynaecology, Faculty of Medicine, Ramathibodi Hospital, 270 Rama 6 Rd., Ratchatawee, Bangkok 10400, Thailand. Email: chonthicha_lek@yahoo.com bs_bs_banner doi:10.1111/jog.12130 J. Obstet. Gynaecol. Res. Vol. 40, No. 1: 96–101, January 2014 96 © 2013 The Authors Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology
  2. 2. overall intrauterine health.14 Endometrial thickness was also shown to positively correlate with receptive- ness. Previous studies reported that thicker endometri- ums give higher rates for IVF and CC-intrauterine insemination (IUI) outcome.14–17 Presently, one of the most effective ways to improve endometrium thinning caused by CC treatment is to add an exogenous estrogens treatment during the course of CC stimulation. However, the effectiveness of exogenous estrogens for the treatment of endometrium thinning during CC cycle still varies. For example, several studies showed that the anti- estrogenic effect of CC on the endometrium might be overcome by the adjunctive use of exogenous estrogens,15–18 while other studies showed that the addition of some types of exogenous estrogens did not significantly enhance the endometrial thickness.19,20 These different outcomes could be the result of several factors, such as the type, dosage, and route of admin- istration of the exogenous estrogens used for the treatment.16,18,19 Estradiol valerate (EV) is an oral synthetic form of estradiol. EV is normally used to condition the uterus for implantation of fertilized ovum, and to promote the maturation and maintenance of female auxiliary repro- ductive organs. Because the oral form of estrogen is more convenient and is relatively safe to use when compared to the transdermal or the vaginal forms, many IVF clinics prefer the oral form of EV in several endometrial preparation procedures.21,22 Here, we studied the effect of EV on the endometrial thickness and folliculogenesis when used in conjunction with CC for ovarian stimulation. Methods Volunteers Thirty-four healthy women aged 20–39 years with regular menstrual cycles of 26–35 days and normal pelvic examinations were recruited in this study. The inclusion criteria were: body mass index 18–25 kg/m2 , day-3 follicle-stimulating hormone (FSH) < 10 IU/L, normal ovulatory cycle which was documented by cycle regularity of previous 3 months, existence of dominant follicle, mid-luteal progesterone Ն4 ng/ml in the recent cycle, and no pregnancy desired. They were advised to use barrier contraception during the study periods, if tubal resection had not been per- formed. Participants were excluded if they had pre- existing ovarian cysts, or had used oral contraceptive pills, gonadotrophin-releasing hormone (GnRH) ago- nists, or depot medroxyprogesterone acetate/GnRH agonist depots within 1, 3 or 6 months of enrollment, respectively. Study design This study was designed as a crossover study, in which the same group of participants would receive the same treatments. Participants were asked for consent and screened for eligibility. The study was involved in four consecutive menstrual cycles. During the first natural cycle, all participants had to be verified to undergo a normal cycle in order to proceed to the next step. Blood samples were taken from all participants for day-3 FSH levels during the first cycle. If the day-3 FSH level of a participant was within the normal range, she would then undergo transvaginal sonographic examination using a 7.5-mHz transducer. The transvaginal sono- graphic examination began on day 12 of the first cycle onwards to determine the ovulation. After 1 week of ovulation, progesterone levels were confirmed. These data were used as the baseline value. After the inclusion and exclusion criteria, all partici- pants were randomly assigned into two groups, based on numbers randomly generated by computers. One group received CC + placebo (vitamin B complex), while the other group received CC + EV in their second cycle. The randomization of group assignment was per- formed by an independent research assistant. The CC + placebo group was treated with CC (100 mg/day) on day 2 to day 6 and then with placebo (vitamin B complex) three tablets/day on day 10 to day 14. The CC + EV group was treated with CC (100 mg/day) on day 2 to day 6 and then with EV (2 mg/tab) three tablets/day on day 10 to day 14. During the second cycle, transvaginal ultrasonography was performed from day 12 until ovulation to document the numbers and sizes of developing follicles, the presence of tri- laminar layer, and the thickness of endometrium. The study was conducted in a double-blinded way so that both the investigator and the participants were unaware of the participants’ group assignment. The third cycle was used as a washout period. The cycle interval was monitored during this period. In the last cycle, both groups were treated again with the same protocols used during the second cycle. Failure to ovulate during any treatment cycle was diagnosed if no leading follicle was larger than 12 mm on day 16 of the cycle, and/or the leading follicle was smaller than 18 mm and was not progressing for at least 3 days. This study was approved by the ethical committee of Ramathibodi Hospital (MURA2007/169). EV improves endometrium on CC cycles © 2013 The Authors 97 Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology
  3. 3. Transvaginal sonographic examination After aligning the uterus along with the central longi- tudinal axis, the endometrial thickness was mea- sured from the echogenic interface between the endometrium and myometrium to the opposite inter- face at the point of maximal thickness.23 The follicular diameter was determined by calculating the mean of the two perpendicular diameters measured at the greatest plane of the follicle. An ovulation was con- firmed by documenting at least two of the following four parameters: a decrease in follicular diameter, blur- ring of the follicular border, the presence of internal echoes, and free fluid in the cul-de-sac.24 Statistical analysis All of the outcomes were analyzed by using stata version 9. The results of descriptive characteristics of the study population are expressed as mean Ϯ standard deviation (SD) for normally distributed data while median and range were used for non-parametric data. The statistical analysis was performed by the Wil- coxon signed rank test for non-parametric data and paired t-test for normally distributed data. P < 0.05 was considered statistically significant. Results Among the 34 women recruited for this study, 32 women met the inclusion criteria. Thirty out of 32 women completed the study, because two women dropped out due to unpleasant side-effects (headache) during the course of CC treatment and thus were excluded from the study. The characteristics of the 30 participants completing the study were as follows: average age 32 Ϯ 4.6 years (range 21–39 years), body mass index 20.4 Ϯ 2.3 kg/m2 (range 15.2–24.1 kg/m2 ), 30 Ϯ 1.5 days of cycle inter- val (range 26–34 days), and the median parity was 1.5 (range 0–3). All participants had normal range of day-3 FSH serum levels with an average at 5.9 Ϯ 1.8 IU/L (range 3.1–8.9 IU/L). The mean mid-luteal progester- one serum level was 14.4 Ϯ 6.2 ng/ml (range 6.2– 27 ng/ml). Table 1 showed the baseline of endometrial thickness in natural cycles was 9.52 Ϯ 1.33 mm, and all of the endometrial patterns were triple layer. The ovulation day was in day 16 (range 13–18) with only one mature follicle in each person per cycle and the maximum follicular diameter was 20.8 Ϯ 1.5 mm. In the first and third cycles, we found that the day of ovulation, the number of mature follicles, the maximum follicle diameter, and the endometrial thick- ness were not different between the groups (data not shown). Consistent with a previous report, CC treatment slightly reduced the endometrial thickness when com- pared with that during the natural cycle (baseline value) (9.04 Ϯ 1.78 mm vs 9.52 Ϯ 1.33 mm, respectively). Notably, EV treatment together with CC significantly enhanced endometrial thickness when compared to that in participants receiving CC + placebo (10.77 Ϯ 1.65 mm vs 9.04 Ϯ 1.78 mm, P < 0.001). We found that, although statistically significant, EV only mildly increased the thickness of endometrium. However, when we categorized the participants who received CC treatment into two groups consisting of the first group, those whose endometrium suffered the endometrium thinning by the CC treatment (endometrium thickness Յ8 mm [n = 12, the average endometrium thickness = 7.4 Ϯ 0.25 mm]), and the second group, those whose endometrium thickness was not affected by the CC (endometrium thickness >8 mm [n = 18, the average endometrium thickness = 10.13 mm Ϯ 1.34]), we found that EV greatly improved those whose endometrium thinned by CC (the average thickness increased from 7.4 Ϯ 0.25 mm to 10.48 Ϯ 0.43 mm, P < 0.0001) (Table 2). On the other hand, EV did not increase endometrium thickness of the second group (10.13 Ϯ 1.34 mm vs 10.97 Ϯ 1.76 mm, P = 0.118). This data suggest that EV treatment is especially beneficial to those who suffer from CC treatment, but may have only a small effect on those with normal endometrium thick- ness. When compared to the baseline endometrium thickness, that of the EV + CC-treated group was slightly thicker (9.52 Ϯ 1.33 mm vs 10.77 Ϯ 1.65, respec- tively, P < 0.001). The endometrial patterns after CC + EV cycles were similar to that after CC + placebo cycles (data not shown). In addition, days of ovulation, numbers of intermediate follicles, numbers of mature follicles, and Table 1 Baseline outcome in natural cycles Outcome Natural cycle Day of ovulation (cycle day)† 16 (13–18) Number of follicles >18 mm 1 Maximum follicle diameter (mm)‡ 20.8 Ϯ 1.5 Endometrial thickness (mm)‡ 9.52 Ϯ 1.33 Endometrial pattern Triple layer 30 (100%) Non-triple layer 0 †Values are given as mean (range). ‡Values are given as mean Ϯ standard deviation. C. Satirapod et al. 98 © 2013 The Authors Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology
  4. 4. the maximum follicular diameters were comparable in both placebo and EV added groups in CC-stimulated cycles (Table 3). Adverse effects We found a few mild adverse effects from participants in the CC + placebo cycles; two dropped out because of headache and one had minimal headache, two partici- pants had dizziness, and two participants had breast tenderness. In the CC + EV cycles, we also observed some mild adverse effects; two participants had dizzi- ness, and three participants had breast tenderness. The rates of adverse effects from both groups were not significantly different (data not shown). No other serious adverse effects were found. Almost all partici- pants with mild adverse effects could tolerate them and completed the study protocol. Discussion Several studies have demonstrated the benefits of adding exogenous estrogen treatment into the CC-stimulated cycle.16–18 These benefits include increased endometrial thickness, improved pregnancy rate, and reduced miscarriage rate.16 Here, we show that administration of EV, an oral form of estradiol, relieved the endometrial thinning effect of CC. Our data suggested that the 6 mg daily regimen of EV for 5 days was sufficient to increase the endometrial thick- ness after CC treatment in normal ovulatory women. Moreover, EV can overcome the effect of CC on the endometrial thickness, especially in patients who have a thin endometrium (below 8 mm). An addition of EV into the CC cycle did not alter the overall pattern of endometrium and yielded the similar tri-laminar pattern observed from the CC + placebo group, therefore retaining the desirable pattern that favors pregnancy.25–27 Exogenous estrogen exerts its positive effect on endometrium by binding to estrogen receptors in endometrium and facilitates endometrium develop- ment.27 It is known that the exogenous estrogen can antagonize the negative effects of CC on endometrial glandular-stromal tissue.17 It has also been shown that 0.05 mg/day18 or 0.02 mg/day17 of oral ethinyl estra- diol for at least 5 days/cycle can prevent the loss of glandular density and increase vacuolated cells during CC stimulation.16–18 We found that the oral form of EV treatment signifi- cantly improved the thickness of the CC-stimulated cycle without interfering with the CC-stimulated tri- laminar pattern, folliculogenesis, and ovulation. Of note, in our protocol, EV was designed to be given on day 10 to day 14 of the cycle, which should not interfere with the effect of CC on FSH levels. The adverse effects found during the CC stimulation were minor and well tolerated. An addition of EV into the CC cycle did not increase any of these adverse Table 2 Effects of subgroup of endometrium thickness between CC and CC + EV Outcome CC + placebo CC + EV P Endometrial thickness† Յ8 mm (n = 12) 7.4 Ϯ 0.25 10.48 Ϯ 0.43 <0.0001 Endometrial thickness† >8 mm (n = 18) 10.13 Ϯ 1.34 10.97 Ϯ 1.76 0.118 †Values are given as mean Ϯ standard deviation. CC, clomiphene citrate; EV, estradiol valerate. Table 3 Effects of ovarian stimulation between CC and CC + EV Outcome CC + Placebo (n = 30) CC + EV (n = 30) P Day of ovulation (cycle day)† 15 (13–18) 16 (13–19) 0.66 Number of follicles Ն18 mm‡ 2.5 Ϯ 1.3 2.2 Ϯ 1.0 0.25 Number of intermediate follicles‡ 2.1 Ϯ 1.2 2.3 Ϯ 1.1 0.37 Maximum follicle diameter‡ 24.9 Ϯ 2.8 25.0 Ϯ 3.7 0.92 Endometrial thickness‡ 9.04 Ϯ 1.78 10.77 Ϯ 1.65 <0.001 Endometrial pattern§ 22 (73.37%) 20 (66.67%) 0.3 †Values are given as mean (range). ‡Values are given as mean Ϯ standard deviation. §Values are given as number (percentage). CC, clomiphene citrate; EV, estradiol valerate. EV improves endometrium on CC cycles © 2013 The Authors 99 Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology
  5. 5. effects, or produce new adverse effects that were not observed in CC stimulation alone. Therefore, adding the oral form of estradiol, EV, to CC cycles could be an effective way to prevent CC-related endometrium thinning with minimum adverse effects. Because of its efficacy and safety, we propose that EV could be used as the estrogen form of choice to prevent endometrium thinning caused by CC stimulation. However, a limitation of this study is that we are only able to show the benefit of EV in normo- ovulatory women and the endometrial thickness is our main outcome. Although, the endometrium thickness is generally well accepted as one of the strong indica- tive parameters for fertility, other parameters should also be investigated. Hence, we suggest that future study may be evaluated in the infertile patient and mea- sured final outcome (pregnancy rate or live birth rate). Given that the thinning endometrium is a major side- effect of CC treatment,9,14,28,29 we envision that EV would enhance pregnancy rate in CC-IUI cycles. We concluded that the addition of EV at 6 mg/day to the CC-stimulated ovulation regimen can significantly improve endometrial thickness in normo-ovulatory women and does not interfere with folliculogenesis. Acknowledgment This study was supported by a Research Grant (No. 50048) from the Faculty of Medicine, Ramathibodi Hos- pital, Mahidol University. Disclosure No author has any potential conflict of interest. References 1. Thessaloniki ESHRE/ASRM-sponsored PCOS Consensus Workshop Group. Consensus on infertility treatment related to polycystic ovary syndrome. Hum Reprod 2008; 23: 462–477. 2. Greenblatt RB. Chemical induction of ovulation. Fertil Steril 1961; 12: 402–404. 3. Badawy A, Elnashar A, Totongy M. Clomiphene citrate or aromatase inhibitors for superovulation in women with unex- plained infertility undergoing intrauterine insemination: A prospective randomized trial. Fertil Steril 2009; 92: 1355–1359. 4. Homburg R. Clomiphene citrate–end of an era? A mini- review. Hum Reprod 2005; 20: 2043–2051. 5. Fiedler K, Ludwig M. Use of clomiphene citrate in in vitro fertilization (IVF) and IVF/intracytoplasmic sperm injection cycles. Fertil Steril 2003; 80: 1521–1523. 6. Lin YH, Seow KM, Hsieh BC et al. Application of GnRH antagonist in combination with clomiphene citrate and hMG for patients with exaggerated ovarian response in previous IVF/ICSI cycles. J Assist Reprod Genet 2007; 24: 331– 336. 7. Acharya U, Irvine DS, Hamilton MP, Templeton AA. The effect of three anti-oestrogen drugs on cervical mucus quality and in-vitro sperm-cervical mucus interaction in ovulatory women. Hum Reprod 1993; 8: 437–441. 8. Marchini M, Dorta M, Bombelli F et al. Effects of clomiphene citrate on cervical mucus: Analysis of some influencing factors. Int J Fertil 1989; 34: 154–159. 9. Dickey RP, Olar TT, Taylor SN, Curole DN, Matulich EM. Relationship of endometrial thickness and pattern to fecun- dity in ovulation induction cycles: Effect of clomiphene citrate alone and with human menopausal gonadotropin. Fertil Steril 1993; 59: 756–760. 10. Haritha S, Rajagopalan G. Follicular growth, endometrial thickness, and serum estradiol levels in spontaneous and clomiphene citrate-induced cycles. Int J Gynaecol Obstet 2003; 81: 287–292. 11. Sereepapong W, Suwajanakorn S, Triratanachat S et al. Effects of clomiphene citrate on the endometrium of regularly cycling women. Fertil Steril 2000; 73: 287– 291. 12. Martinez G, Okai T, Masuda H, Kawamoto E, Yano T, Mizuno M. Transvaginal sonographic assessment of the endometrium in spontaneous and induced cycles. Asia Oceania J Obstet Gynaecol 1990; 16: 239–246. 13. Nakamura Y, Ono M, Yoshida Y, Sugino N, Ueda K, Kato H. Effects of clomiphene citrate on the endometrial thickness and echogenic pattern of the endometrium. Fertil Steril 1997; 67: 256–260. 14. Esmailzadeh S, Faramarzi M. Endometrial thickness and pregnancy outcome after intrauterine insemination. Fertil Steril 2007; 88: 432–437. 15. Elkind-Hirsch KE, Phillips K, Bello SM, McNicho M, de Ziegler D. Sequential hormonal supplementation with vaginal estradiol and progesterone gel corrects the effect of clomiphene on the endometrium in oligo-ovulatory women. Hum Reprod 2002; 17: 295–298. 16. Gerli S, Gholami H, Manna C, Di Frega AS, Vitiello C, Unfer V. Use of ethinyl estradiol to reverse the antiestrogenic effects of clomiphene citrate in patients undergoing intrauterine insemination: A comparative, randomized study. Fertil Steril 2000; 73: 85–89. 17. Unfer V, Costabile L, Gerli S, Papaleo E, Marelli G, Di Renzo GC. Low dose of ethinyl estradiol can reverse the antiestro- genic effects of clomiphene citrate on endometrium. Gynecol Obstet Invest 2001; 51: 120–123. 18. Yagel S, Ben-Chetrit A, Anteby E, Zacut D, Hochner-Celnikier D, Ron M. The effect of ethinyl estradiol on endometrial thickness and uterine volume during ovulation induction by clomiphene citrate. Fertil Steril 1992; 57: 33–36. 19. Ben-Ami M, Geslevich Y, Matilsky M, Battino S, Weiner E, Shalev E. Exogenous estrogen therapy concurrent with clo- miphene citrate–lack of effect on serum sex hormone levels and endometrial thickness. Gynecol Obstet Invest 1994; 37: 180–182. 20. Kinay T, Tasci Y, Dilbaz S, Cinar O, Demir B, Haberal A. The relationship between endometrial thickness and pregnancy rates in GnRH antagonist down-regulated ICSI cycles. Gynecol Endocrinol 2010; 26: 833–837. C. Satirapod et al. 100 © 2013 The Authors Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology
  6. 6. 21. El-Toukhy T, Taylor A, Khalaf Y et al. Pituitary suppression in ultrasound-monitored frozen embryo replacement cycles. A randomised study. Hum Reprod 2004; 19: 874–879. 22. Gelbaya TA, Nardo LG, Hunter HR et al. Cryopreserved- thawed embryo transfer in natural or down-regulated hor- monally controlled cycles: A retrospective study. Fertil Steril 2006; 85: 603–609. 23. Bakos O, Lundkvist O, Wide L, Bergh T. Ultrasonographical and hormonal description of the normal ovulatory menstrual cycle. Acta Obstet Gynecol Scand 1994; 73: 790–796. 24. Ecochard R, Marret H, Rabilloud M et al. Sensitivity and specificity of ultrasound indices of ovulation in spontaneous cycles. Eur J Obstet Gynecol Reprod Biol 2000; 91: 59–64. 25. Bohrer MK, Hock DL, Rhoads GG, Kemmann E. Sonographic assessment of endometrial pattern and thickness in patients treated with human menopausal gonadotropins. Fertil Steril 1996; 66: 244–247. 26. Hock DL, Bohrer MK, Ananth CV, Kemmann E. Sonographic assessment of endometrial pattern and thickness in patients treated with clomiphene citrate, human menopausal gona- dotropins, and intrauterine insemination. Fertil Steril 1997; 68: 242–245. 27. Oliveira JB, Baruffi RL, Mauri AL, Petersen CG, Borges MC, Franco JG, Jr. Endometrial ultrasonography as a predictor of pregnancy in an in-vitro fertilization programme after ovarian stimulation and gonadotrophin-releasing hormone and gonadotrophins. Hum Reprod 1997; 12: 2515–2518. 28. Abdalla HI, Brooks AA, Johnson MR, Kirkland A, Thomas A, Studd JW. Endometrial thickness: A predictor of implanta- tion in ovum recipients? Hum Reprod 1994; 9: 363–365. 29. El-Toukhy T, Coomarasamy A, Khairy M et al. The relation- ship between endometrial thickness and outcome of medi- cated frozen embryo replacement cycles. Fertil Steril 2008; 89: 832–839. EV improves endometrium on CC cycles © 2013 The Authors 101 Journal of Obstetrics and Gynaecology Research © 2013 Japan Society of Obstetrics and Gynecology