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Effects of Clomiphene Citrate Plus Estradiol Or Progesterone On International Journal of Reproductive BioMedicine Volume 18, Issue no. 3, https://doi.org/10.18502/ijrm.v18i3.6718 Production and Hosting by Knowledge E Research Article Effects of clomiphene citrate plus estradiol or progesterone on endometrial ultrastructure: An RCT Robabeh Taheripanah1 M.D., Maryam Kabir-Salmani1, 2 Ph.D., Masoomeh Favayedi1 M.D., Marzieh Zamaniyan3, 4 M.D., Narges Malih5 M.D., Anahita Taheripanah6 B.Sc. 1Infertility and Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2Department of Biomaterials and Tissue Engineering, Stem Cell Division, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran. 3Department of Obstetrics and Gynecology, Infertility Center, Mazandaran University of Medical Sciences, Sari, Iran. 4Diabetes Research Center, Mazandaran University of Medical Sciences, Sari, Iran. 5Social Determinants of Health Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 6Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran. Corresponding Author: Marzieh Zamaniyan; Infertility Abstract Center, Imam Hospital, Amir Background: Pinopods concentrations in endometrial surface is a marker of Mazandarani Ave., Sari, Iran. implantation. Estradiol valerate (EV) was used to change the adverse effects of Postal Code: 4815659769 Clomiphene Citrate (CC) on the endometrium. Tel: (+98) 9113566650 Objective: The goal was to assess whether there is a significant difference in the Email: endometrial pinopods concentrations and other parameters after adding EV and [email protected] progesterone to higher doses of CC. Materials and Methods: In this prospective randomized clinical trial, a total of 30 Received 11 April 2018 women who did not respond to 100 mg of CC from February 2016 to June 2016 were Revised 6 October 2018 evaluated. They were divided into three groups: group I) received 150 mg of CC alone, Accepted 15 October 2019 group II) CC with EV, and group III) CC plus progesterone. On day 21 of the menstrual cycle, endometrial biopsy, a blood sampling, and a scanning by electron microscopy were performed. Production and Hosting by Results: On day 21 of the menstrual cycle, there was no significant difference in the Knowledge E pinopods concentrations (p = 0.641) and serum estrogen levels (p = 0.276) between Taheripanah et al. This groups. However, the Serum progesterone levels in group I was higher than the other article is distributed under two groups (p = 0.007) in the same day. the terms of the Creative Conclusion: Since the addition of EV and progesterone to higher dosages of CC did Commons Attribution License, not change the pinopods concentration and serum estrogen levels on day 21 of the which permits unrestricted menstrual cycle, and the serum progesterone levels was higher in CC alone group (i.e. use and redistribution group I) compared to other groups, it can be concluded that the anti-estrogenic effects provided that the original of CC just appear on the endometrium and not on the plasma levels. author and source are credited. Key words: Ovulation induction, Clomiphene, Estradiol, Progesterone, Electron Editor-in-Chief: microscopy, Endometrium. Aflatoonian Abbas M.D. Registration ID in IRCT: IRCT2015011920408N3. This articel extracted from M.D. thesis (Masoomeh Favayedi) How to cite this article: Taheripanah R, Kabir-Salmani M, Favayedi M, Zamaniyan M, Malih N, Taheripanah A. “Effects of clomiphene citrate plus estradiol or progesterone on endometrial ultrastructure: An RCT,” Int J Reprod BioMed 2020; 18: 201–208. https://doi.org/10.18502/ijrm.v18i3.6718 Page 201 International Journal of Reproductive BioMedicine Taheripanah et al. 1. Introduction apical surface of the endometrial epithelium. These organelles are some micrometers Induction of ovulation is one of the first steps wide and protrude into the uterine cavity of infertility treatment among infertile couples over the microvilli level. Electron microscopy who have polycystic ovarian syndrome, or other is the main instrument used to illustrate causes of infertility such as unexplained infertility. these organelles. Pinopod expression is Clomiphene citrate (CC) is a known and proven restricted to a short-term period of maximal first-line treatment for ovulation induction (1). Data two days in the menstrual cycle presumably confirms that ovulation rate with this method is in the implantation window. The pinopod about 80%, but pregnancy rates are about 40% expression pattern all over the menstrual cycle (2). supports their use as markers of implantation. Different factors such as unexplained infertility HOXA-10 is part of a homeobox gene that might cause the lower rate of pregnancy its expression is essential for endometrial after successful induction, but it should be receptiveness to blastocyst attachment. However, noted that anti-estrogenic effects of CC on it has a crucial role in pinopod growth (8). the endometrium can lead to disturbances in Undeniably, suppressing HOXA-10 leads to endometrium during implantation. This effect reduction in the number of pinopods. In some is dose-dependent and further increases in study, CC leads to a decrease in HOXA- dosage makes this problem more visible 10 and may cause lower pregnancy rate (3). Estrogen can cause endometrial cell (9). proliferation and also increase the cells surface Therefore, in this research, we tried to examine microvillus, change the cell volume, and increase pinopod concentration in the CC-treated group progesterone receptors; this can all support the alone compared with adding estradiol or hypothesis regarding the effect of exogenous progesterone to see whether these changes administered estrogen during ovulatory cycle on in pinopod concentrations could be reversed by pregnancy rate (4). Progesterone can cause hormonal intervention. Our hypothesis was that the uterine glands to become wider and adding estradiol valerate (EV) and progesterone more complex and it also might increase their to higher doses of CC could improve endometrial activity (5). To reduce this effect, researchers morphology such as pinopods concentration have used other drugs such as tamoxifen assessed by electron microscopy and other as ovulation-stimulating agent believing it factors affecting implantation, so that the addition might have less anti-estrogen effect on the may improve implantation compared to CC endometrium and less impact on the cervical score alone. (6). In some studies, addition of conjugated 2. Materials and Methods estrogens in the second half of the follicular phase to compensate for the anti-estrogenic 2.1. Patients effect of clomiphene has been evaluated (7), but no study was done at the cellular Thirty infertile patients were randomly divided levels and endometrial morphology. Pinopods into three groups from February to June 2016 (n are bubbles-like projections found on the = 10/each). The inclusion criteria were woman Page 202 https://doi.org/10.18502/ijrm.v18i3.6718 International Journal of Reproductive BioMedicine Clomiphene and endometrial morphology aged ≤ 35 yr and regular menses (25-34 was performed using Novak curette to take days), and unexplained infertility for at least the samples from the anterior fundal section 1 yr. All patients had normal serum levels of of the uterus. We also took blood samples thyroid-stimulating hormone, and prolactin. Also, for the measurement of serum progesterone. all participants had a history of unsuccessful Quantitative evaluation of uterine dome usage of CC 100 mg/day in prior cycles. density using scanning electron microscopy The exclusion criteria were previous in-vitro was performed as previously described (12). fertilization or intra-cytoplasmic sperm injection, In summary, biopsy samples were washed any other causes of infertility such as hyper immediately in phosphate buffered (0.1mol/l, prolactinemia, thyroid causes, endometriosis, PH 7.4) (PBS tablets, USA, MP Company) and ovulatory dysfunction, and uterine factors. The were then transferred to 2.5% Glutaraldehyde flowchart of eligible patients has been shown in (Germany, DSM company) for initial fixation. Figure 1. Then, the samples were transferred to a solution of 1% osmium tetroxide (Germany, Ridel de 2.2. Ultrastructure of pinopods Haen) as secondary fixation for at least 1 hr. Thereafter, the samples were dehydrated in In the present study, three methods of a graded series of ethanol (5, 50, 70, 90, ovulation induction in infertile patients were 99%) (Razi company, Iran), dried by machine used. For the effect of drugs on pinopods (Polaron CPD 7501 system (VG Microtech, concentration and embryo implantation the UK), and then mounted and covered by endometrium was evaluated at the mid-luteal gold in a Bio-Rad SC510 sputter coater (VG phase (time of implantation). The first group Microtech). received 150 mg of CC (Aboraihan Company, Finally, the number of uterine domes in Tehran, Iran) alone on days 5-9 of menstrual experimental and control groups were counted, cycle. The second group in addition to 150 at the same magnification in six random fields. mg of CC on days 5-9 of menstrual cycle Thus, 30 random fields in each group were received EV (Aboraihan Company, Tehran, assessed morphologically and statistically Iran) for five days from day 8 of menstrual trying to obtain samples from similar regions cycle at a dose of 4 mg daily. In the third of uterus (anterior-fundal wall
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