{"paper_id":"83f72437-033c-415c-a7f0-2d8e6896d0bf","body_text":"The success of a treatment in assisted reproductive technique (ART) cycles depends on\nthe perfect synchrony between embryonic development and endometrial receptivity. The\nimplantation process requires endometrial growth and differentiation of endometrial\nstromal cells. Human endometrium contains growth factors, receptors for growth\nfactors, cytokines, and other key factors for correct embryonic and endometrial\ndevelopment. Multiple embryos fail to implant, and a relevant percentage of IVF/ICSI\ntreatment failures are due to endometrial receptivity disorders ( Blois  et al ., 2011 ;  Farimani  et al ., 2017 ).\nRecurrent implantation failure (RIF) is a common concern among researchers,\nphysicians and patients. Couples desperately require further diagnostic\ninvestigations and/or the use of adjunctive/alternative treatments in order to\nimprove their pregnancy likelihoods. In fact, RIF has been the target of a wide\nscientific debate and several approaches have been developed in order to solve this\nreproduction issue ( Bos-Mikich  et\nal ., 2019 ). Endometrial injury, intrauterine human chorionic\ngonadotrophin (hCG), endometrial receptivity array (ERA), Preimplantation genetic\ntesting for aneuploidy (PGT-A), “Omics” tools (Genomics, Transcriptomics,\nProteomics, Metabolomics), and other techniques have been used in patients with RIF,\nbut the results found still require further analyses for routine use in IVF/ICSI\ncycles ( Somigliana  et al .,\n2018 ;  Nardo  et al .,\n2015 ;  Spencer  et al. ,\n2016 ;  Macklon, 2017 ;  Hviid & Macklon, 2017 . The granulocyte\ncolony-stimulating factor (G-CSF) and Platelet-rich Plasma (PRP) are among these new\ntherapeutic approaches for RIF.\nPRP is prepared from fresh whole blood and contains several growth factors and\ncytokines, including vascular endothelial growth factor (VEGF), transforming growth\nfactor (TGF), platelet-derived growth factor (PDGF) and epidermal growth factor\n(EGF). Therefore, it may help regulate endometrial cell migration, attachment,\nproliferation, differentiation, and neoangiogenesis, resulting in beneficial effects\non endometrial receptivity. The use of Platelet-rich Plasma (PRP) in human\nreproduction is growing, and it could be a new tool to improve clinical outcomes in\npatients undergoing ART procedures ( Zeyneloglu &\nOnalan, 2014 ;  Chang  et\nal ., 2015 ;  Magdi  et\nal ., 2017 ). PRP has been used in assisted reproduction,\nespecially in patients with thin endometria ( Dhillon\n et al ., 2012 ;  Lee\n et al ., 2013 ).\nG-CSF is a hematopoietic lineage-specific cytokine, associated with cell\nproliferation and differentiation, produced by reproductive tissue cells. This\ncytokine promotes endometrial immunomodulation and optimizes the interaction between\nthe embryo and the endometrium. Studies have shown that its use is associated with\nhigher pregnancy rates and lower miscarriage rates ( Lédée  et al ., 2013 ;  Rahmati  et al ., 2015 ;  Arefi  et al ., 2018 ).\nConsidering the beneficial effects of PRP and G-CSF on endometrial receptivity, in\naddition to the fact that failures in IVF/ICSI cycles are related to endometrial\ndisorders, we conducted a pilot study to evaluated if a new therapeutic Protocol for\nImprovement of Endometrial Receptivity (PRIMER) based on the use of PRP and G-CSF\ntogether can improve ART outcomes in patients with RIF.\n\nA total of 66 patients enrolled in the IVF/ICSI program at the Prof Franco Jr.\nCenter for Human Reproduction, from February 2017 to October 2017, and they were\nprospectively included in this study. We obtained a complete medical/surgical\nhistory from all patients, and total screening was performed to exclude RIF\ncauses (a normal karyotype for her and her partner, and no evidence of uterine\ndefects, ultrasonographic evidence of hydrosalpinx, infections, endocrine\nproblems, coagulation defects, thrombophilia and autoimmune defects). We\nconsidered only one fresh embryo transfer cycle. We excluded transfer cycles of\nfrozen embryos.\nThe women were divided into two groups:\n-PRIMER/RIF group: patients with RIF in which intrauterine PRP injection and\nsubcutaneous G-CSF injection were performed. RIF was defined as ≥2embryo\ntransfers (ET), and at least 5 good-morphological embryos were transferred.\n-Control group: patients in their first IVF/ICSI cycle attempt (without PRP or\nG-CSF).\nThe two groups were matched using the Key Performance Indicators Score ( Franco Jr  et al ., 2017 )\nbased on female-age, anti-Müllerian hormone (AMH) levels, number of\nmetaphase-II oocytes, fertilization rates, and morphological quality of\nembryos-transferred.\nAll patients included in the study were submitted to the same ovarian stimulation\nprotocol: the long gonadotropin releasing hormone (GnRH) agonist protocol\n(GnRH-a) as previously described ( Oliveira\n et al ., 2012 ). The starting FSH dose was based\non the patient's age, anti-Müllerian hormone level and antral follicle count\n(Ovarian Response Prediction Index calculation) ( Oliveira  et al ., 2012 ).\nTo induce final oocyte maturation we administered 250µg of recombinant\nhuman chorionic gonadotropin (r-hCG; Ovidrel; Serono, Brazil) subcutaneously\nwhen at least two follicles reached a mean diameter of ≥17mm. GnRH-a was\nadministered until the day of the r-hCG injection. Oocytes were retrieved by a\ntransvaginal aspiration under ultrasound guidance, 34-36 hours following the\nr-hCG injection.\nAll metaphase II oocytes received ICSI, which was carried out as previously\ndescribed ( Mauri  et al .,\n2010 ;  Oliveira  et\nal ., 2011 ). The oocytes were examined after 17-20h to assess\nfertilization; zygotes with two distinct equal-sized pronuclei were considered\nnormal.\nThe embryos were routinely transferred after 96h in culture, and supernumerary\nembryos were cryopreserved. The embryos were then transferred with a Frydman\ncatheter (Frydman ®  Classic Catheter 4.5 CCD Laboratoire C.C.D;\nParis, France) guided by abdominal ultrasound, using a 3.5-MHz convex transducer\n(Aloka SSD-1100; Aloka Co. Ltd, Tokyo, Japan). A single physician performed all\nembryo transfers, and only easy transfers (i.e. the catheter passed smoothly\nthrough the cervix without the need for uterine fixation clamps) with clear\nvisualization of the catheter tip upon ultrasound were considered. All the\npatients received luteal phase supplementation with vaginal natural progesterone\n(Utrogestan ® ; Besins Healthcare, São Paulo,\nBrazil).\nPRP was prepared using autologous fresh whole blood and a double-spin method. In\nbrief, blood was obtained by venipuncture in syringes containing acid citrate\ndextrose (ACD-A) solution (1:4 vol/vol). The citrated blood was centrifuged for\n12 min at 1400 rpm. Subsequently, the supernatant plasma was taken up using a\nmicropipette and transferred into another sterile tube for centrifugation. A\nsecond round of centrifugation was performed for 7 min at 3200 rpm. Platelets\npellet formed at the bottom of the tube. The supernatant was removed in another\nsterile tube and the platelets were suspended in a minimum quantity of plasma by\ngently shaking the tube. The process increases platelet-concentration in 2 to 4\nfold.\nAll patients undergoing the PRP-treatment (study group) received 0.7ml of it\nthrough intrauterine injection, using a soft catheter, 48 hours before the\nET.\nOn the same day of the PRP injection, we injected G-CSF\n(Filgrastim ® , Biosintetica/Achê,\n300µg/0.5ml) subcutaneously, and repeated it weekly. If pregnancy occurs,\nG-CSF is maintained until the 12 th  gestation week.\nThe primary endpoints were ongoing pregnancy and live birth rates. The secondary\nendpoints included implantation, clinical pregnancy and miscarriage rates.\nData management and univariate analysis were carried out using the StatsDirect\nstatistical software version 2.7.9 software (Cheshire, UK). The following\nparameters were evaluated: the woman's age, infertility etiology, number of\noocytes retrieved, number of oocytes in metaphase II retrieved fertilization\nrate, the number of embryos transferred, embryo implantation rates, miscarriage\nrates, ongoing pregnancy rates and live birth rates. We used the nonparametric\nMann-Whitney test to compare the means of continuous variables, when the\ncontinuous variables were not normally distributed, and the Student's t-test was\nused if the continuous variables were normally distributed. The results are\nexpressed as the arithmetic means ± standard deviation (SD). For\ncategorical variables, we used the Fisher's exact test to check between group\nassociations, and the results were expressed as percentages. A\n p  value < 0.05 was considered statistically\nsignificant.\n\nWe found an equal distribution ( p >0.05) of the general and\ncycle's characteristics for PRIMER/RIF (PRP injection and subcutaneous G-CSF\ninjection) and Control (without PRP or G-CSF) groups.  Table 1  summarizes the data.\nGeneral and cycles’ characteristics\nThere were no significant differences between the PRIMER and Control groups regarding\nimplantation, pregnancy, spontaneous miscarriage, ongoing pregnancy or live birth\nrates  p >0.05).  Table 2 \nshows the main results.\nMain results\n\nOur results indicated that the PRIMER enabled patients with RIF (mean number of\nprevious ET of 4.0±1.5) to reach similar ongoing pregnancy and live birth\nrates to those patients who had their first IVF/ICSI cycle attempt, demonstrating\npossible beneficial effects on endometrial receptivity. Unfortunately, to the best\nof our knowledge, the present study is the first to analyze the efficacy of\nintrauterine PRP associated with subcutaneous G-CSF for RIF patients, and thus\ncannot be compared with other results. However, PRP and G-CSF have been employed\nsingly in several medical fields, including human reproduction, with promising\noutcomes, corroborating our findings.\nPrevious trials have shown the beneficial effects of PRP use on tissues, promoting\ncell-differentiation, proliferation, growth, and neoangiogenesis ( Aghajanova  et al ., 2016 ). These\neffects are probably because it contains and/or stimulates several growth factors\nsuch as TGF-β, PDGF, IGF, VEGF, EGF and FGF-2 and bioactive-cytokines, which\nstimulate the inflammatory cascade and the healing process ( Le  et al ., 2019 ). These PRP positive effects\ncould improve reproductive outcomes in patients with impaired endometrial\nreceptivity. However, few trials have evaluated the effectiveness of autologous PRP\nin patients with endometrium disorders ( Chang\n et al ., 2018 ;  Tandulwadkar  et al ., 2017 ;  Zadehmodarres  et al ., 2017 ;  Eftekhar  et al.,  2018 ;  Molina  et al ., 2018 ;  Mehrafza  et al ., 2019 ).\nRegarding the use of G-CSF, studies have shown that its use could improve endometrial\nthickness and pregnancy rates in patients presenting suboptimal endometria ( Jain  et al ., 2018 ). G-CSF is a\nglycoprotein, secreted from endothelial cells, macrophages and some other immune\nsystem cells. Recently, it has been used in assisted reproduction for patients\npresenting thin endometrium and/or recurrent pregnancy loss. Some studies have\ndemonstrated that the G-CSF use by transvaginal endometrial perfusion or\nsubcutaneous way could improve ART outcomes, such as implantation and miscarriage\nrates ( Würfel  et al .,\n2010 ;  Zafardoust  et al .,\n2017 ;  Zhang  et al .,\n2018 ). However, the results in the general population undergoing ART are\nquestionable ( Barad  et al .,\n2014 ;  Jain  et al .,\n2018 ).\nConsidering PRP is obtained through fresh-whole blood peripheral vein, its\nintrauterine-injection is relatively safe, with low-risk of disease-transmission,\nimmune reactions, and deleterious effects to the patient ( Welte, 2014 ). On the other hand, besides G-CSF beneficial\neffects on reproductive tissues, it is considered safe within the dose and route of\nadministration usually employed ( Jang  et\nal ., 2017 ). Therefore, the combined use of subcutaneous\nG-CSF and intrauterine PRP - PRIMER - should be considered as low risk for IVF/ICSI\npatients. In addition, because of the action of these growth factors and\nbioactive-cytokines, PRIMER could have beneficial effects on endometrial\nreceptivity.\nA limitation of this study is that it is not randomized. However, the KPI-score\n(female-age, anti-Müllerian hormone (AMH) levels, number of metaphase-II\noocytes, fertilization rate, and morphological quality of embryos-transferred) and\ncycles characteristics including male-age, FSH total dose, mean number of oocytes\nretrieved, mean number of embryos-transferred, and etiology of infertility found in\nthe study population were similar between the Study and Control groups. These\nfindings confirm that the groups were adequately matched, minimizing bias in the\nstudy outcomes.\nIn conclusion, our results showed, for the first time, evidence that this therapeutic\nprotocol (PRIMER) could be used as a feasible treatment based on biological\nrationale for patients with RIF, since these patients with about four previous ETs\nachieved ongoing pregnancy rates similar to patients who performed their first\nIVF/ICSI attempt cycle. The use of a rigorous match based on the KPI-score provides\nrelevance to the outcomes found. In order to confirm the results found in this\nstudy, randomized controlled trials in a large population should be carried out.","source_license":"CC-BY-4.0","license_restricted":false}