Executive Summary
The objective was to compare effects of GnRH antagonist and standard long protocol agonist on number, and the quality of Metaphase II oocytes, and on reducing risk of hyperstimulation, in patients who are affected by Polycystic ovarian syndrome. Polycystic ovarian syndrome is a common endocrinopathy of the women who are in reproductive age, which is a first cause of the anovulation. GnRH agonists are used in the IVF to avoid premature LH surge before the retrieval of oocyte. Research findings from this study can be significant to various Centers for Reproductive Medicine, and also to all the institutions that practice ART. Consequently, the results of such a research study will help in choice of the most effective protocol in terms of number and quality of metaphase II (MII) oocytes alongside the reduction of hyperstimulation risk. A retrospective study was conducted in Center for Reproductive Medicine X, Sweden. One hundred and seven women affected by Polycystic ovarian syndrome. The measurements that were investigated in the study include; age, type of fertility, BMI, experienced previous stimulation cycles, and ART protocols, ovarian cautelization, duration of infertility, co-morbidity, quality of sperm, last stimulation cycle, Fertilization rate and OHSS incidence. The information on the mentioned measurements was collected from the data base in Centre for Reproductive Medicine X, Sweden. From the findings of this study, there was no statistical significance difference between GnRH antagonist and standard long protocol agonist; hence the hypothesis was accepted.
Abbreviations
PCOS: Polycystic Ovary Syndrome
IVF: In Vitro Fertilization
COH: Controlled Ovarian Hyperstimulation
OHSS: ovarian hyperstimulation syndrome)
GnRH: Gonadotropin-releasing hormone
ART: Assisted Reproductive Technology
LH: Lutenizing Hormone
FSH: Follicle Stimulating Hormone
MBH: medial basal hypothalamus
BMI: Body Mass Index
HCG: Human Chorionic Gonadotrophin
GnRH-R: Gonadotropin-releasing hormone Receptor
CC – clomiphene citrate
ICSI – intracytoplasmatic sperm injection
Introduction
Polycystic ovarian syndrome is a common endocrinopathy of the women who are in reproductive age, which is a first cause of the anovulation. GnRH agonists are used in the IVF to avoid premature LH surge before the retrieval of oocyte. However, patients who are affected by Polycystic ovarian syndrome standard long agonist protocol is bound to be cancelled often because of the multifollicular development, and OHSS (Ovarian Hyperstimulation Syndrome) risk (Tummon 2005). Gonadotrophin antagonist blocks GnRH receptors, competitively, resulting to immediate suppressive effect on the secretion of gonadotrophin (Ron-EL et al. 2000; Felberbaum & Diedrich 1999). As a result, finer modulation of the stimulation of hormones is allowed, as well as reduction of the duration of treatment, which can be from weeks to days (The ganirelix dose-finding study group, 1999).Use of antagonist on patients who have Polycystic ovarian syndrome can result to low incidence of the excessive multifollicular development, ovarian hyperstimulation syndrome, and cancellation of the cycle.
Ovarian Stimulation protocols
The very first IVF treatment, which was successful, was carried out in spontaneous natural cycle (Steptoe and Edwards 1978). Later on stimulation of the ovary became inherent IVF treatment part. There are various protocols and drugs that are used in the IVF treatment. There are long and short protocols. Long protocol has superior efficacy and is more convenient (Marcus 2012). Long protocol is started at the 21st day of cycle preceding the IVF cycle or second day of treatment cycle. The short protocol is carried out by administering GnRH agonist from the second day of treatment cycle and then it is continued to the end of HCG.
Longest and deepest ovaries suppression is usually done using long protocol. Ovaries are suppressed before stimulation is done to ensure some eggs do not develop so quickly, such that all eggs get ready at the same time. Suppression also makes it easy to schedule the treatments. This long protocol involves use of birth-control pills for several weeks, which are overlapped with agonist that is more suppressive, which makes the ovaries well suppressed (Marcus 2012).
However, it is not everyone who uses the long protocol. In particular, women who have low BMI, older patients, women who have ever responded poorly to long protocol, and women whose FSH levels are raised when their periods begins, short protocol is used (Marcus 2012). In the case of antagonist protocol, birth control pill is used to suppress ovaries a bit, and when the cycle starts, GnRH Antagonist is used to prevent the ovulation. This Antagonist protocol is better for the patients with fewer side effects and injections (Hannam, n.d).
The objective of this study was to compare effects of GnRH antagonist, and standard long protocol agonist on number, and the quality of Metaphase II oocytes, and on reducing risk of hyperstimulation, in patients who are affected by Polycystic ovarian syndrome.
Literature Review
Gonadotropin-releasing hormone (GnRH), also called lutenizing hormone-releasing hormone (LHRH), is a reproductive hormone that maintains the functionality and physiology all mammals (Wu, Pagano and Mani 2009: 293). Synthetic GnRH is used in the treatment of various diseases or abnormalities of the reproductive system including endometriosis, prostate cancer (Dong et al. 2011: 457), breast cancer, ovarian cancer (So et al. 2008) uterine fibroids and infertility among others (Millar et al. 2008). The use of GnRH aids in the regulation of the reproductive hormone and its consequent effects thereby correcting the disorders. Gonadotropin-releasing hormone analogs are used to prevent luteinizing hormone from premature surge during COH of infertile couples seeking in vitro fertilization (Fernard 1999). This is the principle focus of this paper and forms the basis of this review.
The decapeptide GnRH, also known as gonadorelin, is synthesized in the hypothalamus, anterior hypothalamus and in specialized neurons within the arcuate nucleus of the MBH (Millar 2005: 6). GnRH is then transported to the median eminence, via axons, where it gets into the hypophysical blood portal system. The “pulse generator”, which is within the MBH, controls the release of GnRH when getting into the hypothysical blood portal system (Skinner et al. 2009 & Dong et al. 2011: 456). GnRH is released once in between 60 and 100 minutes in human beings, the pulsatile release varying with the stage of the reproductive cycle of women (Tannenbaum et al. 2007). GnRH then binds to G-protein-coupled receptor (GnRH-R) that is localized on the gonadotropin cells’ plasma membrane and located within the anterior pituitary stimulating synthesis. This is then followed by release of FSH, LH and gonadotropins (Vien, Leo and Billy 2008: 5458).
Research in GnRH has led to the identification of a number of GnRH isoforms and receptors. The two prominent GnRH isoforms include type-I and type-II each of which has a distinct responsibility and functionality in mammals (Vien, Leo and Billy 2008). Type-I GnRH isoform, for instance, regulates gonadotropins while type-II GnRH isoform, which is a neuromodulator, is responsible for stimulating the sexual behavior in mammals (Hapgood et al. 2005). Similarly, there are two receptors of the GnRH, type-I and type-II of which type-II receptor is not as prominent as type-I receptor in human beings (basically all mammals) (Millar et al. 2004 & Chen and Fernald 2008). Essentially, type-I receptor of the GnRH is a G-protein coupled receptor although it does not have a carboxyl terminal tail, a characteristic that is paramount to the proper functioning of type-I receptor. According to (Millar 2005), type-II GnRH receptor can bind and activate type-I receptor, which is the functional GnRH receptor in mammals, just like type-I receptor can do (Chen and Fernald 2008).
As aforementioned, type-I and type-II GnRH receptors can bind and activate type-I receptor, the functional GnRH receptor in human beings. In doing so, activating, phospholipae C (PhL-C) enzyme cascade is stimulated. Consequently, this leads to accelerated formation of diacylglycerol (DAG) and inositol-triphosphate (IP3). Enhanced formation of IP3 in turn leads to calcium mobilization and consequent activation of protein Kinase C (Boime, Garcia-Campayo and Hsueh 2004). The peak of elevated formation of C (Phl-C) leads to the synthesis and consequent release of FSH, LH and gonadotropins (Cheung and Wong 2008). As aforementioned, GnRH is released at a frequency of once in every 60-100 minutes, the frequency that controls the release of FSH, LH and gonadotropins (Ascoli and Puett 2009). Further control is provided by androgens and estrogen feedbacks (Boime, Garcia-Campayo and Hsueh 2004). Research shows that rate of release of GnRH has a profound impact on the release of FSH and LH and the availability of GnRH-Rs. Low rate of GnRH release increases FSH as it results to high increase of the hormone while high rate of GnRH release increases LH through the stimulation of hormone release (Millar 2005).
The pattern of releasing GnRH, pulsatile, leads to rapid recycling back to the cell surface, internalization and dimerization, which in turn stimulates up-regulation of GnRH receptors (Ascoli and Puett 2009). On the other hand, continuous exposure to GnRH, through increased rate of release of GnRH, down-regulates GnRH-Rs and, consequently, suppresses gonadotropins. This forms the principle of operation of the use of GnRH agonists in controlling ovarian stimulation (Ascoli and Puett 2009). In other words, control of ovarian stimulation through use if the GnRH agonists works on the basis of down regulation of GnRH-Rs through continued exposure to GnRH, which leads to the suppression of gonadotropins.
GnRH agonists lead to the suppression of FSH and LH, by the pituitary gland, while stimulating the development of follicle cells with exogenous gonadotropins. This process came into existence in 1984 when it was first demonstrated (Porter et al. 1984), and it remains to be the fundamental procedure although it has undergone slight, progressive modifications. The procedure remains to be the main protocol used to prevent premature surge of LH in patients undergoing COH for IVF treatment. A number of advantages of GnRH agonists advantages have been identified that make it the primary protocol for the prevention of premature surge of LH. The main advantage, when compared to endogenous GnRH, is that GnRH agonists are more robust than endogenous GnRH because they are less susceptible to destruction (breakdown) by enzymes and the binding efficiency to GnRH-R is significantly high (Millar 2005). On the contrary, endogenous GnRH has a short half life, which ranges between 2 and 4 minutes (Millar 2005) implying they are not robust.
The potency of GnRH agonists is high owing glycine substitution at position 6 with D-amino acids, such as D-Ser, found in Buserelin, and D-Leu, found in Lupron (Millar 2005). Further, the affinity for GnRH-R can be increased, up to ten times, if C-terminal glycinamide is replaced with ethylamide group (Vien, Leo and Billy 2008). All the changes, when combined, has the effect of improving the effectiveness GnRH agonists up to 200 times more than endogenous GnRH, which further explains the reason behind prominent use of GnHR agonists because they are highly efficient in suppressing pituitary gonadotropins by desensitizing gonadotroph cells.
GnRH antagonists are also used as an alternative remedial in place of GnRH agonists whereby they prevent premature surge of LH during COH via an opposing mechanism. When binding to the GnRH-R, antagonists do not induce gonatropin synthesis because they inhibit endogenous GnRH from activation GnRH receptor (Millar et al. 2008). This is entirely opposite in the case of agonists because agonists induce gonatropin synthesis during the binding process. Consequently, antagonists provide an advantage when compared to agonists through the competitive inhibition aspect during binding, which makes the effects of anagonists to be reversible and immediate (Vien, Leo and Billy 2008). Antagonists are characterized by lack of hydrophobic D-amino acids and positions 2 and 3 of which research shows contributes towards the effectiveness of antagonists in generating antagonistic effects (Millar et al. 2008). Further, the N-terminal region is modified when compared to agonists, which has profound effects on the actions of GnRH (Millar et al. 2008).
There are differences between the two protocols with respect to the timing of GnRH analog treatment as well as duration of the same (Vien, Leo and Billy 2008). A GnRH agonist dose is administered on a daily basis from after-menses until oocyte matures, which is triggered by hCG (Vien, Leo and Billy 2008). Stimulation with FSH starts with the suppression by low LH, estradiol (E2) and progesterone, which as about ten days after the patient receives the first GnRH agonist dose. On the other hand, suppression does not go in hand with the initiation of stimulation with FSH in the case of GnRH antagonists. Instead, administration of daily doses of GnRH anatagonists, aimed at inhibiting premature LH surge, is done when the leading follicle has grown to about 14mm in diameter, which happens to be approximately six days after stimulation with FSH occurs. The daily dose continues until oocytes are triggered for maturation (Millar et al. 2008).
Different studies on antagonist long protocols have come up with various findings. GnRH antagonist protocols are associated with reduced requirement for gonadotropin dose (Zikopoulous et al. 2005), lack of flare-induced effects, such as deprivation of sleep, headaches and hot flashes, reduced stimulation duration (Olivennes et al. 2000; Engel et al. 2002; Engel et al. 2003 & Bahceci et al. 2005) and reduction in the risk of OHSS (Hohmann, Macklon and Fauser 2003) when compared to agonist protocols. Other studies have found no statistical significant difference between short and long protocols especially with respect to pregnancy rates (Fluker et al. 2001 & Barmat et al. 2005). On the other extreme end, some studies have found considerable, though small and statistically insignificant, advantage of agonist protocols over antagonist protocols (Albano et al. 2000 & Roulier et al. 2003).
Al-Inany and Aboulghar (2002) attempted to find a way out of the discrepancy that existed in literature, and their study ruled in the advantage of GnRH agonists with respect to pregnancy rates. Similar conclusion was given by Al-Inany et al. (2007). On the other hand, Ludwig, Katalinic and Diedrich (2001) & Daya (2005) found no significant differences between short and long protocols in respect to pregnancy results after treatment. This further adds to the existing discrepancy on agonist and antagonist protocols that needs further research in the aim to come up with a conclusive comparison.
The Research Problem
The research problem gives the research study some sense, which serves to drive the research study (Kumar 2005: 16). There are several protocols that are used in IVF. Some of these protocols could have different effects on the patient’s body. It is important to know the effect of antagonist protocol, and that of agonist protocol to be able to administer the most appropriate protocol in a patient.
Purpose of the Research
This study aimed at comparing effects of Gonadotropin-releasing hormone (GnRH) – antagonist, and standard long agonist protocol among women affected by Polycystic ovarian syndrome.
General objective of the study
The objective was to compare effects of GnRH antagonist, and standard long protocol agonist on number, and the quality of Metaphase II oocytes, and on reducing risk of hyperstimulation, in patients who are affected by Polycystic ovarian syndrome.
Specific objectives of the study
Research Questions
- Do Gonadotropin - releasing hormone antagonist and standard long protocol agonist affect the quality of Metaphase II oocytes in patients who are affected by Polycystic ovarian syndrome?
- Do Gonadotropin - releasing hormone antagonist and standard long protocol agonist affect the quality of Metaphase II oocytes in patients who are affected by Polycystic ovarian syndrome?
- Do Gonadotropin - releasing hormone antagonist and standard long protocol agonist reduce the risk of hyperstimulation in patients who are affected by Polycystic ovarian syndrome?
Research Hypothesis
There is no difference between the use of GnRh antagonist, and the use of a standard long agonist protocol in patients affected by PCOS.
Rationale of the Study
Research findings from this study can be significant to various Centers for Reproductive Medicine and all the institutions that practice ART. The findings revealed whether patients who had used GnRH antagonist and those who had used standard long protocol group had differences in the parameters that were being investigated. Consequently, the results of such a research study will help in choice of the most effective protocol in terms of number and quality of metaphase II (MII) oocytes alongside the reduction of hyperstimulation risk.
Limitation of the Study
This study used secondary sources of information to do the research, which may not be reliable because it may be obtained from unreliable sources, and is prone to error during compiling.
Delimitation of the Study
This study only used secondary data that was obtained from Centre for Reproductive Medicine X, Sweden on the study samples. It also used large samples to ensure that reliability was enhanced.
Research Methodology
This section analyzes how the study was conducted in terms of study population, sample population, sampling strategy procedures, study design and sampling. It also has the validity and reliability of the secondary sources, measurements and ethical consideration which are crucial elements of consideration n a research study (Welford, Murphy and Casey 2012: 32).
Study Population
The study population of this study was the patients affected by PCOS who had attended Centre for Reproductive Medicine X, Sweden.
Sample Population and Sampling Strategy
In this retrospective study, 107 women, who were affected by PCOS, were sused. Those women were selected non-randomly. The non random sampling made it possible to identify the patients who participated in the study using information that was available in them, relevant to the retrospective study (Miles and Huber 2004).
Research Design and Sampling
Retrospective study was applied to compare the GnRH agonist and standard long protocol agonist. The research used secondary data that was collected from the databases in Centre for Reproductive Medicine X, Sweden The study sample were the IVF Patients with PCOS, who had attended Centre for Reproductive Medicine X, Sweden. The study included 107 IVF patients. Simple random sampling was employed, while selecting the 107 women.
Quantitative research specifies numerical assignment to the problem being investigated. It was used in this study because findings that were collected from the samples were accurate reflection of the total population that the samples had been drawn from. In addition, large sample was used, making it fairly confident that figures reflected the status of the population the sample was representing, and was statistically valid (VanderStoep and Ohnson 2009).
Validity and Reliability of the Secondary Sources
Information on study subjects was obtained, from the relevant database in the Centre for Reproductive Medicine X, Sweden only, and many parameters were investigated. The different types of parameters helped in constructing validity.
Measurements
The measurements that were investigated in the study include; age, type of fertility, BMI, experienced previous stimulation cycles, and ART protocols, ovarian cautelization, duration of infertility, co-morbidity, quality of sperm, last stimulation cycle, Fertilization rate and OHSS incidence. The information on the mentioned measurements was collected from the data base in Centre for Reproductive Medicine X, Sweden.
Data Analysis
Data obtained from secondary sources was analyzed quantitatively by summarizing all the key findings statistically. The data analysis was done within the selected samples and across the selected samples.
Ethical Consideration
Permission was obtained from the Centre for Reproductive Medicine X, Sweden department of medical records. The data that was collected in the study was analyzed professionally such that obtained data was not interfered with to favor the research. The obtained information was used for the purpose of this research only. All sources of the secondary data that were used in the study were acknowledged.
Results of last stimulation cycle
No statistically significant differences were observed between cycles nor in number of folicules, oocytes retrieved, nor in number of fertilized oocytes or embryo transfers. Summary of results are showed in Table No.5.
Fertilization rate was 42.1% (n=16) in long protocol group and 49.3 % (n=34) in short protocol group, that did not reach the level os statistical significance (Chi-square, p=0.48).
Discussion
It was investigated that mean age of patients who were in the long protocol group was (31.13 SD± 4.06), while the mean age of patients who were in short protocol group was (29.35 SD± 3.46). This shows that there was no statistical significance difference in age between short protocol group and long protocol group.
With respect to the type of fertility, 58 patients had primary infertility, while 49 patients had secondary fertility. It was found out that 44 (63.8%) patients, who had primary infertility, were in short protocol group while 14 patients with primary infertility were in long protocol group. Twenty five patients (36.2%) who had secondary infertility were in short protocol group, while 24 patients (63.2%) with secondary infertility were in long protocol group. These findings show that short protocols were mostly used in patients with primary infertility, while long protocol were mostly used in patients with secondary infertility.
In reference to BMI, patients in long protocol group had BMI mean of 23.18, while patients in short protocol group had BMI mean of 22.62. This shows that there is no significant differences, at 0.05 level of significant, between the BMI of women in short protocol groups and that of women in long protocol group. These research findings were similar with the research findings of Ashrafi et al (2005).
Analysis of patients who had experienced previous stimulation cycles, and ART protocols was done. It was found out 90 women had experienced previous stimulation cycles and ART protocols. Among the women who were in short protocol group, 19 (50%) had had experience with clomophene citrate, 15 (39.5%) had had in vitro fertilization, while 4 (10.5%) had had intracytoplasmatic sperm injection. Women who were in the long protocol group, 31 (44.5%) had had experience with clomophene citrate, 15 (21.7%) had had in vitro fertilization, while 10(14.5%) had had intracytoplasmatic sperm injection.
Previous ovarian cautelization was done in 14 (36.8%) women who were in short protocol group, and in 28 (40.58%) women in long protocol group. Analysis was done using Chi-square, where p=0.43 meaning that there was no statistical significance difference, at 0.05 level of significant,between women in the short protocol group and those in long protocol group. These research findings were similar with the research findings of Ashrafi et al (2005).
The mean duration of infertility was calculated in both groups using Mann-Whitney test. It was found out that mean duration of infertility among women in long protocol group was 3.82 (SD 1.72) years, while that of women in short protocol group was 3.96 (SD 2.94) years. This means that there is no statistical significance difference, at 0.05 level of significant, between the duration of infertility in women in long protocol group, and those in short protocol group.
Serious co-morbidity was analyzed among 48 women; 13(37.1%) in short protocol, and 35 (52.2%) in long protocol group. The women in both protocol groups reported at least one serious co-morbidity. The results were analyzed using Chi-square and it was found out that there is no statistical significance difference, at 0.5 level of significant, between serious co-morbidity reported by women in the short protocol group, and those in long protocol group.
The quality of sperm was compared in short protocol group and in long protocol group and the results analyzed. Student-T test results indicated that there was significantly lower A+B sperm count in the short protocol group [44.25 (SD 15.4)] than in long protocol group [50.55 (SD 11.6)]. These findings show that there was statistical significance difference, at 0.5 level of significant, in A+B sperm count between the short protocol group and long protocol group.
Through the Mann-Whitney test, it was found out that there was a higher percentage of morphologically normal sperm in short protocol group [15.92.25 (SD 16.42)], than in long protocol group [9.97 (SD 10.9)]. These findings show that there was statistical significance difference, at 0.5 level of significant, in A+B sperm count between the short protocol group and long protocol group. The concentration of sperms was analysed in the two groups using Mann-Whitnney test; and it was found out that There was no significant different between short protocol groups’ sperm concentration [54.36 (SD 54.0)], and long protocol groups’ [58.8 (SD 41.6)]. These findings show that there was statistical significance difference, at 0.5 level of significant, in sperm concentration between the short protocol group and long protocol group.
The description of the last stimulation cycle was done, analyses done statistically, and results presented in table 5. Ovitrelle had been used to trigger 48 (69%) women in short protocol group, and 29 women (80.6%) women in long protocol group. These findings show that most patients were triggred with Ovitrelle. Diferilin had been used to trigger 19(27.5%) women in short protocol group, and 5 women in long protocol group. Pregnyl had been used to trigger 2 women in short protocol group, and 2 women in long protocol group. The mean day of use of Cetratide, in short protocol group was 7.7 (SD 1.03), and its mean duration of use was 4.7 (SD 1.2) days.
The findings of the last stimulation were summarized and presented in table 4. There were no statistically significant differences, at 0.05 level of significant, that were observed between the cycles, in number of folicules, in number of fertilized oocytes, or in embryo transfers. These research findings were in line with Minaretzis et al. (1995) and Hwang et al. (2004) who found no difference in number of the oocytes retrieved in the antagonist group and in the agonist group. These findings were different from Ashrafi et al (2005) research findings, who found out that Using short protocol GnRH antagonist results in more number of retrieved oocytes, which are of good quality. In their research, Ashrafi et al (2005) found out that the number of Metaphase II in the antagonist group was higher than those in standard long agonist group.
Fertilization rate of 16 women in long protocol group, and that of 34 women in short protocol group was analyzed using Chi-square. In short protocol group, fertilization rate was 42.1%, while in ling protocol group, fertilization was 49.3%. These findings indicate that there was no statistical significant difference at 0.05 level of significant. These findings were similar to previous research done by Hwang et al. (2004).
OHSS incidence of 15 women in long protocol group, and 25 (36.2%) women in short protocol group were analyzed using Chi-square. The findings were quite similar with (39.5%) in long protocol group, and 36.2% in short protocol group. These findings indicate that there was no statistical significant difference (at 0.05 level of significance) between the OHSS incidence in short protocol group, and OHSS incidence in long protocol group. OHSS stages did not differ between the long protocol group and short protocol group. These findings indicate that there was no difference in OHSS stages in short protocol group and long protocol group. However, these findings differed from Ashrafi et al (2005) research findings that long antagonist protocol is better that antagonist protocol. Hohmann, Macklon and Fauser (2003) also had different research findings whereby they found out that short protocol antagonist reduced in the risk of ovarian hypestimulation syndrome.
Conclusion and Recommendation
The study was successful and GnRH antagonist and standard long agonist protocol were compared among women who were affected by polycystic ovarian syndrome, meeting the objectives of the study. The Use of GnRH antagonist and standard long protocol GnRH agonist was found to be similar, as generally there was no statistical significance difference in both protocol. These research findings made the hypothesis to be accepted since it was stating that there is no difference between the use of GnRh antagonist and the use of a standard long agonist protocol in patients affected by PCOS. The research findings of this study were similar to some research findings of some other researches. It is recommended that this research be done using prospective study, and research be done using the same measurements, to find out whether there would be any difference in the research findings.
