Introduction. Acta Medica Mediterranea, 2018, 34: 1765

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1 Acta Medica Mediterranea, 2018, 34: 1765 THE STUDY OF NATURAL VERSUS HORMONE REPLACEMENT THERAPY CYCLES IN FROZEN EMBRYO TRANSFER IN INFERTILE COUPLES ON PREGNANCY OUTCOME: A DOUBLE BLIND RANDOMIZED CONTROL TRIAL MOJGAN JAVEDANI MASROUR *, FATEMEH ASHTARY Research and Clinical Center of Gynecology and Fertility, Shahid Akbarabadi Hospital, Iran University of Medical Sciences, Tehran, Iran ABSTRACT Background: The present Randomized controlled trial (RCT) was conducted to evaluate reproductive outcomes of frozen embryo transfer (FET) during hormonally intervened cycle and also during physiological condition. Methods: The present RCT was conducted on 200 couples (240 cycle) with male-originated infertility aged 19 to 39 years at the Infertility Clinic of Shahid Akbar Abadi hospital, Tehran. The Participants were randomly allocated into two group; the natural cycle (n=115 cycle) and hormonal cycle (n=125 cycle). The hormonal group was received 4-6 mg of oral estradiol on the third day of their cycles; while controls received placebo. Transvaginal ultrasound was used for evaluation of endometrial thickness and when the thickness was reached to8 mm, embryo transfer was planned. Chemical and clinical pregnancy were considered to be the primary and secondary outcomes, respectively. Results: A total of 28 cycles were cancelled in both groups; 15 cycles (8.3 percent) in the natural group and 13 cycles (7.6 percent) in the hormonal group. Inter-groups statistical analysis showed that serum levels of FSH, LH and estradiol were not statistically different between the study groups (P>0.05). On the other hand, chemical clinical pregnancy rate (hormone receiving group: 32.7% and control group: 33.3%) and clinical pregnancy rate (hormone receiving group: 34.5% and control group: 26.4%) statistically were not different between the groups (P> 0.05). Conclusion: The study results showed there is not any statistical significant difference between the study groups from point of view of chemical and clinical pregnancy rate. There is still a controversy whether one is superior over the other, so further studies are needed. Keywords: Endometrium thickness, Pregnancy outcome, Hormonal cycle, natural cycle. DOI: / _2018_6_270 Received March 30, 2018; Accepted June 20, 2018 Introduction Although the implantation rate has increased over the last 30 years since the onset of infertility treatment by in vitro fertilization (IVF), researchers are still trying to improve the clinical outcome of IVF by improving endometrial receptivity and embryo development (1). It is estimated that 60% of fetuses resulting from IVF/ICSI methods do not implant after transfer. In most cases, the possible cause of implantation failure is reduced endometrial receptivity of the transferred embryo (2). Successful implantation of the embryo in FET requires good-quality embryos, suitable transfer time for endometrial receptivity and efficient interference between the embryo and the receiving endometrium (3). Dynamic changes in the endometrium during natural cycles are controlled physiologically by the body hormones. Administration of exogenous hormones during a cycle can change the endometrial epithelium and have different effects on implantation. In recent years, the number of FET cycles has been more frequently considered than IVF and ICSI because of tendency to transfer fewer embryos,

2 1766 Mojgan Javedani Masrour, Fatemeh Ashtary improved laboratory techniques, as well as easier and less expensive cycles (4, 5). Currently, hormonal factors such as estrogen are used in many cycles for growth and preparation of the endometrium. This is associated with a high fertility rate in some cases; however, there is insufficient information available on the definite influence on the fertility rate and factors associated with it. Studies have shown that it can be hypothesized that the fertility rate will increase if the natural cycles in infertile patients are replaced with regular hormonal cycles. The present study was undertaken to compare the fertility rate between physiological and hormonal cycles as it relates to endometrial preparation. Materials and methods The study design and procedures Women aged years, planning an FET cycle at the Infertility Clinic of Shahid Akbar Abadi hospital were invited to take part in this trial. They were eligible to participate if they were assisted reproductive techniques such as IVF and ICSI with frozen embryo transfer cycles due to male factor, had a regular menstrual cycle, serum levels of follicle stimulating hormone (FSH) less than 10 IU/dl and normal serum prolactin level. Women were excluded from participating in the trial if they were allergic to estradiol or progesterone, had uterine anomaly, polycystic ovary syndrome (PCOS), endometriosis stage III/IV, preimplantation genetic diagnosis cycles, tubal factor and history of receiving donated oocytes. The study protocol was approved by the Research Ethics Committee of Iran University of Medical Sciences (Ethic number: IR.IUMS.REC ), and was registered with before patient recruitment started (IRCT code: IRCT N1). All procedures took place at the Shahid Akbar Abadi hospital Fertility Unit, and patients gave consent to participate in the study between March 2016 and April The design of current study was a double blind RCT comparing the efficacy of natural and HRT cycles in FET treatment. Both treatment protocols were standard protocols in use at the Shahid Akbar Abadi hospital Fertility Unit at the time. After initial screening and fulfilment of inclusion and exclusion criteria, research participants were randomized, using a table of random numbers, to one of two groups: natural FET (n=100, 115cycle) and HRT FET (n=100, 125cycle). All subjects intending to commence the intervention and meeting the study criteria were invited to the fertility unit between days 1 and 3 of their monthly cycle for baseline scan and study enrollment. At the first visit those who wished to take part, completed written consent form and were randomly assigned into mentioned groups. The baseline information for each participant (age, duration of infertility, and the results of ultrasound and laboratory tests) was recorded on data entry forms. The study procedures then followed the unit FET treatment protocols according to the groups. The women in the hormonal group were administered 4-6 mg of estrogen in the form of oral estradiol valerate (Progynova ; Schering, Madrid, Spain) on the third day of their cycles as the intervention after a transvaginal ultrasound. A second transvaginal ultrasound was performed after days of estrogen treatment. If endometrial thickness was at least 8 mm, embryo transfer (Only blastocyst embryos) was planned. Natural micronized progesterone (Utrogestan ; Seid, Madrid, Spain) was vaginally administered at a dose of 400 mg/ 12 h for 3 or 5 complete days before embryo transfer, depending on the cleavage stage of embryos (embryo age +1day). Progesterone supplementation continued if pregnancy occurred until 12 weeks of pregnancy. In participants of natural FET (as control) vaginal ultrasound monitoring was performed after spontaneous menstruation. They were taken 4-6 mg of estrogen placebo in the form of oral on the third day of cycle. Then they had an ultrasound assessment between days 10 and 12 of their cycle to confirm endometrial thickness and follicular growth. Additional ultrasound monitoring was done in subsequent days if necessary. Based on the ultrasound examinations, when the mean diameter of the leading follicle was at least 14 mm, patients were taught to start self-monitoring of ovulation using urinary dipsticks for the endogenous LH surge. After LH Surge, when LH began to fall, it actually showed ovulation. The minimal endometrial thickness had to be 8 mm to perform FET. Only blastocyte embryos were transferred 7 days after LH surge. In order to assess the LH levels correctly, determination should be performed at least daily, and preferably twice a day.

3 The study of natural versus hormone replacement therapy cycles in frozen embryo transfer In the present study, chemical pregnancy (based on hormone levels of β-hcg) was considered as the primary outcome and clinical pregnancy (existing fetal heartbeat) was considered as the secondary outcome. Sample size calculation On the basis of sample size formula suggested for randomized clinical trials, the number of subjects needed in each arm with type I error of 5% (α = 0.05) and Type II error of 20% (β = 0.2; power = 80%) is 80. Considering 25% dropout, total number of subjects in each group was calculated as 100. Statistical analysis Data were analyzed using the SPSS software, version 22 (SPSS Inc., Chicago, IL, USA). Quantitative data are presented as mean ± standard deviation (SD), and qualitative data are demonstrated as frequency and percent. The Kolmogorov-Smirnov test was used to assess the normality of data. Inter-groups statistical analysis was done using Independent Sample T test and chi-square for quantitative and qualitative variables, respectively. Results were considered statistically significant at p-value less than Figure 1: flowchart of inclusion and exclusion of trial participants. As shown in table 1, inter-group statistical analysis revealed no meaningful changes in terms of serum levels of LH, FSH and estradiol (P>0.05). Endometrial thickness significantly increased (P= 0.03) in HRT receiving group (9.65 ± 2.21mm) compare with placebo receiving controls (9.01 ± 1.92 mm). Results Baseline characteristics of the study subjects are reported in table 1. A total of 240 cycles from 200 infertile couple due to male factor were recruited in the current RCT. Mean age of participants in hormone replacement therapy (HRT) receiving group was 31.5± 5.9 and 33.5± 6.1 was mean age of subjects in control group; the difference between groups was not significant (P= 0.430). The HRT FET and control groups included 125 and 115cycles, respectively. The cancellation rates were 8.3 percent (15 cycles) and7.6 percent (13 cycles) in HRT FET and control groups, respectively (Fig.1). On the other hand, 2 abortions in HRT FET group and 3 abortions in control group was occurred; which, abortion rate was not statistically significant between the study groups (P= 0.47). Also no significant differences were observed between the groups from point of view of infertility type and mean IVF/ICSI, number and quality of embryos transferred (P>0.05). Current study results showed chemical pregnancy occurred in 36 cycles of treatment groups (32.7 percent) and 29 cycles of control ones (33.3 percent). Both chemical and clinical pregnancy rate were not statistically different between the groups (P>0.05). Discussion In the present study there was no significant difference in chemical and clinical pregnancy rate

4 1768 Mojgan Javedani Masrour, Fatemeh Ashtary between participants in the hormonal group and the natural ones. Some findings from other studies are in accordance with our results (6, 7). In a recent study conducted by Ginny et al, on 159 women were randomized (80 Natural; 79 HRT) it s reported that, the outcomes- for women with ovulatory cycles undergoing FET - are similar between natural and HRT protocols (8). In the other study conducted by Tarek et al, on 212 women with regular menstrual cycles, findings suggested that both FET protocols were same effectiveness regarding implantation rate and pregnancy outcome (9). On the other hand, in this field there are some studies with controversial results compare with our findings. For example Morozov et al. reported that HRT reduced the rate of endometrial preparation for pregnancy compared to the natural cycle (10). The researchers introduced changes in the implantation process as the main cause of the reduced pregnancy rate following increased levels of estrogen (11). Yu Zheng et al. reported that the use of HRT increased the fertility rate. Because of the undesirable effect of HRT on the fertility rate, it is recommended be used in the EFT cycle (12). Two other studies found that HRT for endometrial preparation increased the pregnancy rate when compared with the natural cycle (13, 14). On the other hand Levron et al. and Zhuoni et al, reported that under optimal embryo conditions, natural FET cycles had higher implantation and pregnancy rates compared with HRT-based FET cycles (15). In the present study, mean endometrial thickness was significantly higher in the hormonal group than the natural group. In general, endometrial thickness is an effective method for evaluating endometrial development and predicting its receptivity (16). Some researchers consider a minimum endometrial thickness of 6 mm as necessary for successful pregnancy. Confirming this finding, studies have reported that HRT for preparation of an endometrium with an endometrial thickness of greater than 8 mm at the time of embryo transfer increases the pregnancy rate (10, 14). Some studies have reported conflicting results in this regard. For a patient with a thin endometrium under treatment to increase endometrial thickness, estradiol therapy alone was not sufficient to increase endometrial thickness. Another study found that the expression pattern of estrogen receptors was associated with a thin endometrium (17). There are some possible reasons for this controversy and bias in the findings of different studies such as different inclusion and exclusion criteria, mean age of participants, and poor controlled situation. Overall there are some advantages and disadvantages for both natural and HRT methods. Numerous studies have been conducted to evaluate the efficacy of the different protocols of endometrial preparation but still there are controversy among infertility specialists about the most appropriate endometrial preparation protocol (18). On one hand there are several reasons to justify the effectiveness of the use of HRT to increase the fertility rate. Generally, planning for melting and embryo transfer in natural cycles is difficult because of differences in the follicular phase length. The use of HRT, however, allows the day of embryo transfer to be determined by inference and increases the rate of embryo implantation. It is also difficult to identify a positive surge of luteinizing hormone in the ascending and descending stages of natural cycles, which causes difficulty in determining the appropriate day for embryo transfer implantation with natural cycles. In some cases, ovulation may be predictable and might have reached the correct stage, but the endometrial thickness may not be sufficient to prevent pregnancy failure (12). In addition, the increase in the rate of fertility by the use of hormonal preparations as opposed to natural cycles could be because less time is needed to prepare the uterine and endometrial stages of the patients. Hormonal preparation for pregnancy can help determine the exact time of transfer and thawing of the fetus without the difficulty of timing in natural cycles. The use of hormonal preparations can be effective in increasing the pregnancy rate in some patients, such as women over 40 years of age and those who have experienced early menopause or amenorrhea (14, 19). On the other hand, researchers suggested some advantages for natural cycles such as being safe, preferable, and cost-effective protocol for FET. They believe that, whenever possible, the natural endometrial preparation should be preferred. While in patients under prolonged ovarian down regulation, patients with ovulatory dysfunction and ovarian failure, HRT should be applied (20).

5 The study of natural versus hormone replacement therapy cycles in frozen embryo transfer Conclusion In conclusion, in the present study there was no significant difference in chemical and clinical pregnancy rate between patients in the HRT and natural groups although the mean endometrial thickness was significantly higher in the hormonal group than the natural group. Based on the findings of the current study, it seems that none of these two methods are superior to each other and have same success regarding pregnancy rate. However, in order to achieve more accurate results, further studies with larger sample sizes and more controlled situation among the various social levels are needed. References 1) Fox C, Morin S, Jeong JW, Scott RT, Lessey BA. Local and Systemic Factors and Implantation: what is the Evidence? Fertil Steril 2016; 4: ) D Ly K, Aziz N, Safi J, Agarwal A. Evidence-based management of infertile couples with repeated implantation failure following IVF. 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Endometrial receptivity in an in vitro fertilization program as assessed by spiral artery blood flow, endometrial thickness, endometrial volume, and uterine artery blood flow. Fertil Steril 2001; 2: ) Wang A, Ji L, Shang W, et al. Expression of GPR30, ERalpha and ERbeta in endometrium during window of implantation in patients with polycystic ovary syndrome: a pilot study. Gynecol Endocrinol 2011; 4: ) Casper RF, Yanushpolsky EH. Optimal endometrial preparation for frozen embryo transfer cycles: window of implantation and progesterone support. Fertil Steril 2016; 4: ) Queenan JT, Jr., Veeck LL, Seltman HJ, Muasher SJ. Transfer of cryopreserved-thawed pre-embryos in a natural cycle or a programmed cycle with exogenous hormonal replacement yields similar pregnancy results. Fertil Steril 1994; 3: ) Levron J, Yerushalmi GM, Brengauz M, Gat I, Katorza E. Comparison between two protocols for thawed embryo transfer: natural cycle versus exogenous hormone replacement. Gynecol Endocrinol 2014; 7: Acknowledgment The authors would like to thank Iran University of Medical Sciences for supporting this scientific work. Corresponding author Mojgan Javedani Masrour Research and Clinical Center of Gynecology and Fertility, Shahid Akbarabadi Hospital Iran University of Medical Sciences Tehran, Iran Moulavi St., Tehran, Iran javedani46@yahoo.com