Preventing premature luteinizing hormone (LH) surge is a fundamental step during controlled ovarian hyper-stimulation (COH) of IVF/ICSI cycle, which is commonly accomplished using one of the Gonadotropin-releasing hormone (GnRH) analogues, GnRH agonists or GnRH antagonists . Nevertheless, they follow different suppression patterns. GnRH agonists act as indirect suppressors of the pituitary GnRH receptors causing a transient flare-up phase, followed by a down-regulation of GnRH receptors , while GnRH antagonists directly and competitively occupy the GnRH receptors leading to a dose-related inhibition of gonadotropins secretion . However, GnRH receptors are expressed both in pituitary and extra-pituitary tissues, including the ovary , which may allow the GnRH analogues to affect the ovarian function through paracrine/autocrine mechanisms. Nevertheless, it is unclear whether these effects are dependent on the type of GnRH analogues used.
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among reproductive-aged females, with a worldwide prevalence of 5–20% [4, 5]. The principal manifestations of this syndrome are ovulatory dysfunction, hyperandrogenism, and polycystic ovarian morphology . Besides, PCOS accounts for 90–95% of anovulation cases attending infertility clinics . Although the pathophysiology of PCOS is still not fully understood, growing evidence suggests a pivotal role for angiogenic dysregulation . PCOS ovaries exhibit higher vascularization and lower impedance to flow in ovarian stromal vessels compared to control [8–10], which may be arisen from the differences in the levels of ovarian angiogenesis regulators as PCOS women showed higher levels of vascular endothelial growth factor (VEGF) and lower levels of the soluble form of VEGF receptor-1 (sVEGFR-1) compared to control both in serum and follicular fluid samples [11, 12]. In addition, Based on the study of Tal et al. , follicular fluid placental growth factor levels (FF PlGF), unlike serum levels, are increased in PCOS subjects. PlGF and VEGF are angiogenic growth factors belonging to the vascular endothelial growth factor (VEGF) family, which contains VEGF-A (also known as VEGF), VEGF-B, VEGF-C, VEGF-D, and VEGF-E, that is known for its role in regulating vasculogenesis and angiogenesis . On the other hand, the sVEGFR-1 acts as an anti-angiogenic factor by sequestering VEGF and PlGF and decreasing their free form availability . VEGF is considered the fundamental member of the VEGF family, and most of the available researche on angiogenesis were interested in detecting its role more than the other VEGF family members. However, although data are still relatively limited, higher levels of circulating PlGF have been linked to a variety of pathological conditions such as metastatic breast cancer , leukemia , rheumatoid arthritis , systemic lupus erythematosus , metabolic syndrome , type II diabetes , coronary artery disease , and neovascular age-related macular degeneration . In addition, growing evidence has shown an important role of PlGF in regulating the reproduction process, starting from ovulation  to placentation, implantation, and embryo development [25–28]. Consequently, imbalance in PlGF levels may lead to pregnancy complications like preeclampsia, giving birth of small for gestational age, preterm birth, and stillbirth [29–32]. These effects may arise from the pleiotropic effects of PlGF, as besides its proangiogenic activity, PlGF acts as an immune modulator by enhancing monocyte  and macrophages activation , inhibiting dendritic cells differentiation and maturation , and regulating uterine NK cells proliferation and/or differentiation . In addition, it skews the type 1 T helper immune response to the Th2 phenotype . PlGF is expressed in follicular fluids at higher levels compared to circulation, and FF PlGF levels correlate positively with the number of retrieved oocytes of IVF/ICSI cycles . However, no previous study has investigated the impact of the GnRH analogues protocols on the FF PlGF levels either in the general IVF/ICSI population or in the PCOS one.
Anti-Müllerian hormone (AMH), also known as Müllerian inhibiting substance (MIS) or Müllerian inhibiting substance factor (MIF), is a member of the transforming growth factor (TGF)-β family. It was initially recognized for its role in the differentiation of the male reproductive tract by triggering the regression of the Müllerian ducts, the Anlagen of the oviduct, uterus, cervix, and the upper part of the vagina [37, 38]. However, proceeding studies demonstrated its pivotal role in regulation ovarian folliculogenesis during both FSH-independent stages, by inhibiting the recruitment of primordial follicles [39–41]; and FSH-dependent stages, by reducing the follicular sensitivity to FSH [42, 43] and suppressing estradiol (E2) production from the granulosa cells (GCs) [43, 44]. In follicles, GCs are the principal producer of AMH , but the production depends on the follicle development stage. It is initiated at low levels in primary follicles, reaches its highest levels at pre-antral and small antral follicles, and subsequently declines during the following follicular growth stages [46–48]. Elevated AMH levels were observed both in serum and FF samples of PCOS women compared to controls [49–53]. Although the cause of this elevation is still unknown, current evidence suggests that it may be a result of the increased number of pre-antral and small antral follicles seen in PCOS ovaries and the overproduction of AMH per GC [54, 55]. In addition, some studies reported an abnormality in AMH/AMHR regulation system in PCOS subjects [56–59]. Previously, Winkler et al.  demonstrated a reduction in the expression of AMH from GCs after treating them with a GnRH antagonist for 24 hours. On the contrary, the expression was increased from the GnRH agonist-treated GCs models. Similarly, Dong et al.  noticed a down-regulation in the expression of AMH mRNA and AMH protein from the GnRH antagonist-treated GCs, but not from the GnRH agonist-treated GCs or the (GnRH agonist + GnRH-antagonist)-treated GCs. However, in these studies, the cells were treated with the GnRH analogues after they were cultured, and the authors did not mention whether the samples were taken after COH using the GnRH agonist protocol or the GnRH antagonist one. Moreover, it was unclear whether they included/excluded PCOS subjects from the study criteria. To the best of our knowledge, no previous study investigated the effect of GnRH analogues during COH protocols on the follicular fluid levels of AMH (FF AMH) in PCOS subjects.
Selecting the best gametes is crucial for IVF/ICSI success. Thus, mature oocytes would usually be subjected to a morphological assessment before being injected with the chosen sperms using an inverted microscope to evaluate their cytoplasmic maturation and detecting the presence of certain extra and intracytoplasmic morphologic features [62, 63]. However, it is still unclear how the type of GnRH analogue used in the COH protocol would influence the oocyte morphology; since some reports showed an improvement in oocyte quality in the GnRH agonist group compared to the GnRH antagonist one [64, 65], while others support the GnRH antagonist side , and some found no differences between the two groups [66, 67]. Nevertheless, none of these studies focused on the PCOS population.
Considering the previous data, we conducted this trial to compare the effect of the type of the GnRH analogues used in COH protocol on the follicular microenvironment of PCOS women undergoing IVF/ICSI cycles and to clarify whether these effects would have any impacts on the final IVF/ICSI embryological or clinical outcomes.
This clinical trial aimed to compare the effects of the long GnRH agonist protocol and the flexible GnRH antagonist protocol on the follicular fluid levels of PlGF and AMH, clinical and embryological IVF/ICSI outcomes, including oocyte morphology in PCOS subjects.