AIP Conference Proceedings 2193, 040006 (2019); https://doi.org/10.1063/1.5139368 2193, 040006
GnRH receptor expression and endometrial cell proliferation of Macaca nemestrina after administration of GnRH agonist in controlled ovarian stimulation procedures
Cite as: AIP Conference Proceedings 2193, 040006 (2019); https://doi.org/10.1063/1.5139368 Published Online: 10 December 2019
Andhea Pradhita, Nurhuda Sahar, Muharram Natadisastra, et al.
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GnRH Receptor Expression and Endometrial Cell Proliferation of Macaca nemestrina after Administration of
GnRH Agonist in Controlled Ovarian Stimulation Procedures
Andhea Pradhita
1,a), Nurhuda Sahar
2,b), Muharram Natadisastra
3, Wa Ode Zulhulaifah
1, and Rosalina Thuffi
11Master Program Biomedical sciences, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Central Jakarta 10430, Indonesia
2Department of Biology, Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Central Jakarta 10430 Indonesia
3Department Obstetrics and Gynecology, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Central Jakarta 10430 Indonesia
Corresponding author: a)andheadebbypradhita@gmail.com
b)nurhudasahar@yahoo.com
Abstract. GnRH is widely used in the embryo fertilization program (FIV) as one of the ovarian stimulation regimens. At the implantation window, GnRH agonists are known to have an effect on the endometrium directly or indirectly. GnRH estimated has a negative effect on the development of endometrial cells after ovarian stimulation. This study is to analyze the impact of GnRH agonist on ovarian stimulation procedures on receptor expression and endometrial cell apoptosis. The study sample was a stored biological material (BBT) from serum and the endometrial tissue of Macaca nemestrina. The total sample is 8 and divided into 2 groups, the stimulated and control groups. Each sample will be examined 2 types which are the enzyme-linked immunosorbent assay (ELISA) for serum and immunohistochemistry (IHC) for endometrial tissue.
IHC was performed with anti-GnRH and caspase 3 antibody. Concentration is measured using an ELISA reader and then converts Optical Density (OD) to a concentration using SoftMax Pro Software. Calculations of stained and unmarked cells were performed using the Image-J Analyzer programs and scored by HSCORE. There is no significant difference between GnRH serum in stimulation or control group (p=0.773). There was no correlation between GnRH in serum with GnRH receptor in endometrial tissue (p=0.060) and activation of caspase (p=0.193). There was no difference between stimulation group and control group in the amount of GnRH in serum, presence of active GnRHR, and Caspase 3 activation. Keywords:
GnRH receptors, Macaca nemestria, GnRH Agonist, Controlled ovarian stimulation Keywords: Controlled ovarian stimulation, GnRH agonist, GnRH receptors
INTRODUCTION
Development and the human reproductive system are regulated through the hypothalamus-pituitary – gonads axis.(1) Gonadotropin-releasing hormone (GnRH) is the main regulator of production in terms of pituitary gonadotropin release for gonad development and sex steroid production [1]. GnRH release is controlled by the neurotransmitter and neuropeptide input, then transported to the gonadotrope cell in the anterior pituitary. The response of GnRH receptors (GnRHR) in the anterior pituitary stimulates the production of FSH and LH [1,2]. GnRH is widely used in In Vitro Fertilization (IVF) as one of the ovarian stimulation regimens [3,4]. The ovarian stimulation protocol uses a combination of follicle-stimulating hormone (FSH) with Gonadotropin-releasing hormone (GnRH)
both agonists or antagonists, plus oral supplementation, and ovulation triggers [5]. The agonist mechanism is a prolonged response causes a decrease in FSH and LH production. The purpose of using GnRH agonists in ovarian stimulation protocols is to prevent premature LH surges through pituitary gland desensitization [6,7]
Besides the pituitary, GnRH receptors are detected in the epithelial cells of the endometrial gland and stroma with dynamic patterns and a significant increase in the secretion phase [8]. The presence of GnRH receptors in endometrial cells is thought that the GnRH agonist (GnRH Analogue) has a direct or indirect effect on endometrial development during the menstrual cycle [9]. Functionally, GnRH regulates cell proliferation, apoptosis, and remodeling [10].
Several studies have reported that controlled ovarian stimulation has a negative effect on the rate of pregnancy success [11-13]. GnRH analogs (GnRHa) induces apoptosis and reduces cell proliferation in ovarian cancer cell lines, endometrial cancer cell lines, breast cancer cell lines, and also endometriosis [14-16].
Menstrual physiology and histology of the endometrial mucosa are regulated by a balance between cell proliferation and apoptosis for successful implantation of the embryo [17].Activation of peripheral GnRH receptors causes a decrease in intracellular cyclic adenosine monophosphate (cAMP) and activation of the protein tyrosine phosphatase. Tyrosine phosphatase interferes with growth factor-induced tyrosine phosphorylation and with a mitogen-activated protein kinase cascade (MAPK). Decreased regulation of gene transcription in the cell nucleus and increased apoptosis due to decreased cytoplasmic cAMP. Then the MAPK pathway will be reduced. GnRH-induced apoptosis can accelerate luteolysis and accelerate the endometriotic process [18]. Disruption of apoptotic regulation in the endometrium can cause failure of In Vitro Fertilization (IVF) [19]. In this study, an analysis of GnRH receptor expression and endometrial cell proliferation in the endometrial tissue of Macaca nemestrina in the luteal / secretory phase after GnRH agonist was carried out.
MATERIAL AND METHODS Subject
The design of this study is retrospective experiment. The study sample was a stored biological material (BBT) from serum and the endometrial tissue of Macaca nemestrina from the previous study. Animals used in this experimental study are female monkeys (Macaca nemestrina) at reproductive age. The criteria are 8-10 years old, weight 5-8 kg, and have already given birth. The use, procedure for treatment, and handling of Macaca nemestrina has been approved by the institutional animal care & use committee (ACUC) Center for Primate Animal Studies issued by Bogor Agricultural Institute (IPB) Bogor. Animals that have been selected are marked (tattoo number in the groin area) and then inserted into individual cages made of stainless material and placed in a special room. These animals are quarantined or adapted to new individual cages for 2-3 periods of the menstrual cycle. During this period animal health is maintained, treatment if needed, and ad-lib meals.
Total samples are 8 and divided into 2 groups, the stimulated and control groups. The stimulation group was given the long protocol gonadotropin-releasing hormone. There are three regimens used: 1. Gonadotropin / FSH recombinant (Gonal-F), 2. Gonadotropin-releasing hormone / GnRH agonist (Suprefact) and 3. Human Chorionic Gonadotropin / hCG (Pregnyl). GnRH agonist (suprefact) dose of 160 μg starts to be given to the luteal phase in the middle of the previous menstrual cycle and continues until the day before ovulation, for approximately 14 days. After obtaining estradiol hormone levels <70 pg / mL on the second day of menstruation, administration was combined with recombinant FSH (Gonal-F) for three doses, each of 30 IU, 50 IU and 70 IU for each group. Recombinant FSH (Gonal- F) is injected on the second day after menstruation with a dose according to the treatment group for 10-12 days until the peak of estradiol secretion is obtained. hCG dosage of 10,000 IU or equivalent to 3,200 IU of Macaca nemestrina was performed.
We will use 50 IU dose and control to be analyzed. Each sample will be examined 2 types which are the enzyme- linked immunosorbent assay (ELISA) for serum and immunohistochemistry (IHC) for endometrial tissue.
Sample Preparation
Serum
The serum was stored at -84℃. The serum to be examined is at 3 different times (Before Stimulation, After Stimulation, and mid-luteal phase (Day 21) in menstrual cycle. We will use ELISA mid-luteal phase to be analyzed.
Before ELISA began, serum was stored at room temperature.
Endometrial Tissue
Endometrial samples are stored in paraffin blocks. A total of 8 samples endometrial tissue taken at mid-luteal phase (Day 21) in menstrual cycle or estimated after follicular phase of estradiol surge. Slides prepared through tissue from paraffin blocks use microtomes.
ELISA Tested
GnRH concentration was measured from serum Macaca nemestrina using Monkey GnRH ELISA Kit (EMK0135, Finetest) with a sensitivity of <9,375 pg / ml. The principle of the method is the competitive ELISA. First prepare the serum to be examined and standard solutions with various dilutions. Enter the sample and Biotin-Labeled Antibody into the well then incubate for 45 minutes at 37℃. Wash the well. Enter HRP-Streptavidin Conjugate (SABC) then incubate for 30 minutes at 37℃. Wash the well. Enter the TMB substrate followed by incubation at 37℃ in dark within 15-20 minutes. Enter stop solution. Concentration is measured using an ELISA reader and then converts Optical Density (OD) to a concentration using SoftMax Pro Software. The wavelength used is 460 nm.
Immunohistochemical (IHC) Tested (GnRH receptors and Caspase 3) using One-Step, Neopoly Polymer Detection Kit, BioGear.
The 0.4-0.5 µm slices of tissue from the paraffin blocks are fixed to a glass object coated with aminopropyl triethoxysilane (Apex), deparaffinization, and rehydrate tissue section. Rinse tap water. Give endogenous peroxidase blocks then incubate slides for 10-15 minutes. Rinse tap water. Do antigen retrieval step using retrieval step using Retrieval Generation One (RG1). Turn off heat source, when timer is up and allow it to cool. When the pressure has dropped, carefully remove the lid. Lift the dye container and place it on the table. After staining, let the slide stand at room temperature. Rinse several times in phosphate buffer and continue with staining. Provision of primary antibodies and incubation according to the protocol, then rinse 3 times in a buffer. (anti-GnRHR / GnRH receptor antibody (ARG56020; F1G4; Arigo Laboratories; 1:50) and Caspase 3 antibody (GTX30246; CPP32 4-1-18; GeneTex;
1:100) in different slide). Give Universal Polymer HRP then incubate for 20-30 minutes at room temperature. Rinse 3 times in a buffer solution. Add 1 drop (30µl) DAB Chromogen concentrate to each 1 ml DAB Buffer, mix in a circular manner and apply to the network for 5-10 minutes. Rinse with running water. Apply counter stain. Using Hematoxylin Mayer’s and then incubate for 1 minute. Rinse 3 times in distilled water. Apply Bluing Reagent and incubate for 5-10 minutes. Rinse immediately with distilled or deionized water. Slides are dehydrated and cleaned in xylene. Coverslip uses permanent mounting media. The positive control for GnRH receptors used is human pituitary.
The positive control for Caspase 3 is Ca Mammae. Negative control is the endometrial tissue that is not reacted with the marker being examined for all slides.
Assessment of Immunohistochemical
The slides are observed under a light microscope with an objective magnification of 40 times on the 5 fields of view. The slides are photographed by Leica application suite. Calculations of stained cells were performed using the Image-J Analyzer program and IHC profiler. The IHC slide was observed on the intensity of brown color that appears and the percentage of colored cells (on cytoplasm). IHC profiler measures pixel automatically. This value only shows semiquantitative data in the categories. Further calculations are needed to get the results of quantitative analysis, namely by calculating the scoring using the IHC Optical density score method:
Score = [(percentage contribution of high positive x 4) + (percentage contribution of positive x 3) + (percentage contribution of low positive x 2) + (percentage contribution of negative x 1)]/100
Remarks: Score 1: colored weak (negative) Score 2: <5% (weak positive) Score 3: 5 - 50% (positive) Score 4:>
50% (strong positive)
RESULT Elisa tested
In this study, 8 serum samples were tested by ELISA to measure GnRH levels in serum in both groups. The GnRH protein is tested by competitive ELISA where antibodies that later interact with the GnRH protein have been coated in microwells. The basic principle of competitive ELISA is to add a competitor in microwells, in the form of the same antigen that has binding to the enzyme. Antibodies in microwells bind to the antigens examined, in this test GnRH proteins. Then, antibodies that do not bind to the testing protein are bound with the competitor's antigen, GnRH-R, which is already bound to the conjugate HRP. The antibody bond with GnRH-R HRP conjugate then binds to the substrate and shows color. The reading of the staining results is reversed, where the stronger the color, the lower the concentration of the GnRH test.
TABLE 1. Description result of serum Elisa of Macaca nemestrina in midluteal phase Midluteal ELISA GnRH N Mean Rank Sum of Ranks P value
Control 4 4.25 17.00 0.773
Stimulation 4 4.75 19.00
FIGURE 1. Boxplot analyzed from 2 groups. Analyzed using the Mann Whitney Test.
The unit of concentration is in pg / ml.
Based on the test results, the average concentration of GnRH in the control group was 46,004 pg / ml with a minimum value of 18,442 pg / ml and a maximum of 92,896 pg / ml. In the stimulation group, the average GnRH concentration was 68,888 pg / ml with a minimum value of 23,319 pg / ml and a maximum value of 151,184 pg / ml.
There was no significant difference of GnRH concentration in control group and in stimulation group (P-value 0.773).
It was analyzed by Mann-Whitney test.
IHC GnRHR
GnRHR expression is carried out through immunohistochemistry in order to see the location where the GnRH receptor is expressed. Prior to Immunohistochemical testing, Hematoxylin Eosin (HE) was carried out to determine cell morphology and ensure that tissue could still be used for research. GnRHR expression, both in the control group and the stimulation group, was assessed in the glandular epithelium. The results show that the cytoplasm is colored brown on the gland. Positive controls used in this test are from the anterior pituitary tissue of humans.
Figure 2 shows the results of immunohistochemical staining to detect GnRHR expression in glandular epithelial control samples and samples with stimulation. Overall, the results of the outward appearance indicate the presence of GnRHR expression in the cytoplasm.
FIGURE 2. Immunohistochemical staining for GnRHR antibody. (A) Positive control is human pituitary tissue (40x). (B) Stimulation group (40x). (C) Control group (40x). (D) Negative control (10x).
FIGURE 3. Boxplot of Immunohistochemical staining for GnRHR antibody. Analyzed using the Mann Whitney Test.
TABLE 2. Description result of IHC GnRHR Endometrial tissue of Macaca nemestrina in midluteal phase
IHC GnRHR N Mean Rank Sum of Ranks P value
Control 4 5.50 22.00 0.248
Stimulation 4 3.50 14.00
According to the results of the analysis, the mean of the control group's optical density GnRHR score was 1.3638 and the mean of the stimulation group was 1.2425. The average GnRHR optical density score showed that the stimulation group expression was not significantly different than the control (p-value 0.248). It was analyzed by Mann- Whitney test.
IHC Caspase 3
Caspase 3 expression is carried out through immunohistochemistry in order to see the location where the caspase 3 is expressed. Prior to Immunohistochemical testing, Hematoxylin Eosin (HE) was carried out to determine cell morphology and ensure that tissue could still be used for research. Caspase 3 expression, both in the control group and the stimulation group was assessed in the glandular epithelium. The results show that the cytoplasm is colored brown on the gland. Positive controls used in this test are from the Ca Mammae tissue we got from Dharmais Cancer Hospital.
Figure 4 shows the results of immunohistochemical staining to detect Caspase 3 expression in glandular epithelial control samples and samples with stimulation. Overall, the results of the outward appearance indicate the presence of Caspase 3 expression in the cytoplasm.
FIGURE 4. Immunohistochemical staining for Caspase 3. (A) Positive control is Ca Mammae tissue (40x). (B) Stimulation group (40x). (C) Control Group (40x). (D) Negative Control (10x).
FIGURE 5. Boxplot of Immunohistochemical staining for Caspase 3.
TABLE 3. Description result of IHC Caspase 3 Endometrial tissue of Macaca nemestrina in mid-luteal phase
IHC Caspase 3 N Mean Ranks Sum of Ranks P-value
Control 4 4.25 17.00 0.767
Stimulation 4 4.75 19.00
The average Caspase 3 optical density score showed that the stimulation group expression was not significantly different than the control (p-value 0.767). It was analyzed by Mann-Whitney test.
Correlation between Elisa GnRH with IHC GnRHR.
The researcher intends to analyze the relationship between the amount of GnRH in the serum and the number of GnRH receptors in tissue. Table 4 shows that there is no significant correlation between serum GnRH levels and GnRH-R (p-value 0.060). It was analyzed by Pearson Correlation.
TABLE 4. Correlations between GnRH in serum with GnRHR in Endometrial tissue of Macaca nemestrina in mid-luteal phase
Variable N r P value
GnRH 8 -0.595 0.060
GnRHR 8 -0.595 0.060
Correlation between Elisa GnRH with IHC Caspase 3.
The researcher intends to analyze the relationship between the amount of GnRH in the serum and Caspase 3 in tissue. Table 5 shows that there is no significant correlation between serum GnRH levels and Caspase 3 (p-value 0.193). It was analyzed by Pearson Correlation.
TABLE 5. Correlations between GnRH in serum with caspase 3 activation in Endometrial tissue of Macaca nemestrina in mid-luteal phase
Variable N r P value
GnRH 8 -0.356 0.193
Caspase 3 8 -0.356 0.193
Correlation between IHC GnRHR with IHC Caspase 3
The researcher intends to analyze the relationship between GnRHR in tissue and Caspase 3 in tissue. Table 6 shows that there is no significant correlation between GnRHR and Caspase 3 (p-value 0.256). It was analyzed by Pearson Correlation.
TABLE 6. Correlations between GnRHR with caspase 3 activation in Endometrial tissue of Macaca nemestrina in midluteal phase
Variable N r P value
GnRHR 8 0.273 0.256
Caspase 3 8 0.273 0.256
DISCUSSION
Biological materials stored in the form of serum and Macaca nemestrina endometrial tissue were chosen as research samples because they are close to humans in terms of their reproduction systems. The anatomy and physiology of the female reproductive system Macaca nemestrina is similar to the human female reproductive system [20]. Macaca nemestrina females have a menstrual cycle length of 28 to 30 days. In humans, the median period of menstruation is 28 days with a duration of between 21 days to 35 days [21]. The menstrual cycle is important in preparing the receptive endometrium for pregnancy. The implantation process is known to require many factors so that it can occur perfectly. Receptive endometrium is needed which is when the implantation window for blastocyst can attach to the endometrium. Ovarian stimulation in the IVF procedure is known to cause significant changes in endometrial secretion. Long-term exposure to GnRH in the ovarian stimulation protocol can be used as the gold standard for most in vitro fertilization (IVF) strategies. However, only 5% of implantation successes and 15% of clinical pregnancies occur in infertile patients undergoing the GnRH agonist protocol [4].
GnRH is a decapeptide synthesized by specific neurons located in the preoptic region in the hypothalamic arcuate nucleus. GnRH is pulsatilically released into the blood portal. GnRH consists of 10 amino acids and has a similar structure among various species, with at least 50% identity of the same sequence. In addition to the hypothalamus, the expression of GnRH-I (mammalian GnRH) has been shown in somatic and gonadal tissue such as the endometrium, placenta, ovary, fallopian tube, and trophoblast [22]. GnRH agonists (e.g., Buserelin, goserelin, triptorelin, and leuprolide) have similar structures to the original GnRH and can be strongly bound to GnRH receptors. They are also resistant to destructive enzymes and a longer half-life compared to native GnRH (native GnRH has a half-life of about 2 to 4 minutes compared to 3 hours) [15]. Glycine substitution at the position of amino acid no 6 significantly increases plasma half-life compared to native GnRH. Some agonists replace ethyl amide with glycine at the amino acid position No. 10 which can increase the affinity for GnRHR [3,23]. According to table 1 and figure 1, even GnRH amount in stimulated serum is higher than control serum. There are no significant differences between them (p>0.773). Maybe the half-life is not long enough to persist until the mid-luteal phase. So, at that time only native GnRH remains in the stimulation’s group serum.
GnRHR requires stimulation by GnRH to be active. GnRHR stimulation was obtained from natural GnRH or GnRH agonists. The COOH terminal of decapeptide (GnRH) is very important for binding to receptors. While the NH2 terminal ends are important for receptor binding and also for receptor activation [8]. The flare effect of the GnRH agonist treatment requires about 1-3 weeks later to get the hypogonadotropic hypogonadal state [24]. The mechanism underlying homologous desensitization of gonadotrophin secretion stimulated by GnRH in the absence of receptor desensitization is unknown. It is thought to involve post-receptor adaptive processes such as regulation of IP3 receptors and desensitization of voltage-operated Ca2 + channels. In addition, preliminary research on the GnRHR pattern shows that agonists induce their endocytic internalization of the plasma membrane by reducing the amount of cell surface GnRHR, resulting in a reduction in GnRH-induced agonist receptors on the cell surface. The amount of GnRHR can cause desensitization of gonadotrophin secretion that is stimulated by GnRH even in the absence of receptor desensitization [25]. The receptor dimer is internalized after the signal is present and cannot return to the membrane as long as the membrane receptor remains occupied. then the cell is unresponsive to the presence of a GnRH signal [26].The results of the study found that the number of receptors was lower in the stimulation group than in the control group, although the difference is not significant (p>0.248). But there are no data about profile GnRH receptors in endometrium after stimulation procedures in IVF treatment.
Apoptosis that occurs in the endometrial secretory phase occurs normally removing old or dysfunctional cells and to provide tissue repair in each menstrual cycle. Caspase 3 will increase in normal endometrium during the menstrual cycle secretion phase [19]. Li, et al. investigated caspase 3 activity on endometrial cycle, and Apoptosis that occurs
during the advanced secretory phase and the menstrual phase is involved in remodeling that will occur in the endometrium in the transition from the secretion phase to the proliferative phase after menstruation [17]. Beigi, et al.
was investigated incidence of apoptosis in rat endometrium after ovarian stimulation in the implantation period. He said ovarian stimulation could change the expression of several genes associated with apoptosis and increase the incidence of endometrial apoptosis in the implantation period. Then apoptosis can affect implantation and endometrial reception [27]. An excessive dose of stimulation can cause ovarian suppression. Therefore ovarian stimulation can cause luteal phase defects that require supplementation [3]. Luteal defect had increased caspase 3 levels in out-phase endometrium [28].
Park Se Ra et al. Isolated human endometrial stem cells. They examined the response of stem cell proliferation and found a decrease in the rate of growth of endometrial stem cells given GnRH compared to untreated control cells.
GnRH also increases the activity of caspase 3 pro-apoptosis and DNA fragmentation. He also used FSHr as a co- treatment. According to Park Se Ra's hypothesis, exposure to exogenous GnRH can directly damage endometrial stem cells which consequently can reduce pregnancy success with GnRH-based IVF treatment. GnRH agonists suppress the PI3K / Akt signaling cascade, which is involved in many physiological functions, including stem cell proliferation, differentiation, and migration [29]. It can be concluded, GnRH agonists could make luteal phase defects as an indirect effect and decrease proliferation of stem cells in endometrium as a direct effect.
There are no significant difference between 2 groups for caspase 3 activation (p>0.767) from this research. Isabelle Cedrin- Durnerin et al. did a research IVF stimulation using a short protocol GnRH agonist. They said, in spite of a decreased steroid ovarian response to exogenous gonadotropins, other ovarian response and IVF outcomes were similar [30].
This GnRH / GnRHR system regulates a very important process for the local invasion of trophoblasts for implantation of embryos in the endometrium [8]. From the result in table 4, there is no correlation between GnRH serum and GnRH Receptors (p>0.060). We taught it because we took the endometrial sample in mid-luteal phase (around day 21), even though the end of stimulation is around day 14. Maybe the GnRH recombinant is no longer in serum. The rat estrous cycle from another research, an increase in the number of GnRH receptors is seen in the afternoon of the proestrus just before the gonadotropin surge occurs [8,18]
In endometrial carcinoma, uterine leiomyosarcoma, and ovarian carcinoma, peripheral GnRH receptors combine with Gi protein. A decrease in cAMP and reduced activity of the MAPK pathway ultimately decreases the regulation of gene transcription in the cell nucleus. This event mediates the antiproliferative activity of GnRH and its receptors on tumor cells [18]. In table 5, there is no correlation between GnRH serum amount with activation of caspase 3 (p>0.193).
There is no correlation between GnRH receptors and caspase 3 (p>0.256). Another research using testis Gold Fish (Carassius auratus) gave different result, GnRH-induced apoptosis in goldfish testes increases levels of proteins such as fas and fas ligand and increases caspase-3 and caspase -8 activity.(31) Limonta said that the expressed GnRHRs were significantly related to antiproliferative activity [32]. But, they still didn’t know the mechanism.
CONCLUSION
The maturation of endometrium, also the proliferation mechanism, is not the direct result of GnRH and GnRH-R binding mechanism. Hyperstimulation can cause ovarian suppression, which can cause luteal phase defects. There is an experiment that concluded that luteal phase defects could increase caspase 3 levels beyond the natural endometrial cycle. GnRH agonists could make luteal phase defects as an indirect effect and decrease proliferation of stem cells in endometrium as a direct effect. But from this research, we conclude there is no difference between stimulation group and control group in the amount of GnRH in serum, presence of active GnRHR, and Caspase 3 activation. There are other mechanisms that affect the mid-luteal phase of the endometrium to become not receptive or mature at the time of implantation, so pregnancy is difficult in IVF patients.
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