Influence of EmbryoGlue® Transfer Medium on Implantation of Human Embryos

Introduction: Thanks to ever-growing advances in medical science, couples who are in the in vitro fertilisation (IVF) now have more options than ever to encase their chances at a successful pregnancy. One of the options is the use of EmbryoGlue (EG), that creates a bridge between the embryo and the uterus and provides protection to the embryo itself during the transfer process. Aim of this study was to determine whether EG medium is of greater importance for embryo implantation than conventional medium in assisted reproductive technology and compare the rate of embryo implantation with EG and conventional medium in relation to the quality of the embryo, the age of the patients and tobacco smoking. Methods: The retrospective study included 50 patients who used EG medium in embryo transfer (ET) and 50 patients in the control group using conventional medium. All patients underwent ET after stimulation of the cycle according to a short protocol. ETs were done on Day 2, 3, or 5 in the blastocyst stage. Age and smoking status were recorded. Results: Out of a total of 100 patients, 42 patients had successful implantation and positive β-hCG 15 days after ET. In a control group 38 % had positive β-hCG and in the group of patients who used EG 46 %. A higher rate of embryo implantation success was observed on the second day of transfer in the group of patients using EG. In the EG group a significant increase in the embryo implantation rate was observed in patients older than 35. In tobacco smokers the implantation rate was higher if they used EG during ET. Conclusion: EG medium had a positive effect on the second day of ET, patients above the age of 35 and patients who were tobacco smokers.


Introduction
Embryo transfer (ET) is a multi-stage process and one of the most sensitive steps in the in vitro fertilisation (IVF) procedure. From aspiration to transfer, it is necessary to support the oocytes and later, the development of the embryo by providing optimal cultivation conditions. The combination (ingredients) of the transfer medium is considered to be important for the interaction of the endometri-um and the embryo during implantation. 1 After transfer, the embryo must rely on its own ability to implant and use of hyaluronan-enriched media can improve implantation success.
IVF programs continuously seek to increase the rate of implantation and pregnancies. 2 Advances in stimulation regimen and therapy, cultivation

Results a) Implantation success
Out of a total of 100 patients, 42 patients had successful implantation and positive beta human chorionic gonadotropin (β-hCG) 15 days after ET. In a control group (50 patients), 19 had positive β-hCG and 31 negative β-hCG, representing 38 % of embryo implantation success rate. In the group of patients who used EG, 23 patients had positive β-hCG and 27 negative β-hCG, which makes a 46 % success rate. There is an obvious difference in implantation success rate between these two groups of 8 % in favour of EG medium, however, difference was not statistically significant (Chi-squared test, p = 0.418) ( Figure 1).

b) Quality of the embryo
In the control group, the success rate of embryo implantation (positive β-hCG) on the Day 2 was 28.6 % (4 of 14 patients), on the Day 3, 37 % (10 out of 27 patients) and on the Day 5, 55.6 % (5 out of 9 patients) ( Figure 2). media, and technology contribute to this. 3 By designing cultivation media, which mimic changes in the female reproductive tract, it is possible to optimise development and create a healthy environment for the embryo. 4 EmbryoGlue (Vitrolife) ® (EG) is a medium modelled exclusively for ET with a proven effect of increasing implantation. EG has a basic composition of blastocyst culture medium and contains high concentrations of hyaluronan (HA) and recombinant albumin. It can be used to transfer all stages of embryonic development, including blastocysts after assisted hatching, biopsy, and cryopreservation. HA is a glycosaminoglycan naturally present in vivo in follicles, oviducts, uterine fluids and may be involved in the implantation process, 5, 6 by binding to the CD44 glycoprotein receptor of the embryo allowing HA to enter the cell. 7,8 HA is ubiquitous in the body, however, its action at different locations of the reproductive tract critically depends on its size, which is controlled by the balance of synthesis using one of the three isoforms. 9 Due to its similar consistency, HA increases the viscosity of the transfer medium and may enhance the ability of the embryo to interact with uterine fluid. Thanks to these properties, it improves the apposition and binding of embryos, which are key steps in the implantation process. 10,11 Aim of this study was to determine whether EG medium is of greater importance for embryo implantation than conventional medium in assisted reproductive technology (ART) and compare the rate of embryo implantation with EG and conventional medium in relation to the quality of the embryo, the age of the patients and tobacco smoking.
The retrospective study included 50 patients who used EG medium in ET and 50 patients in the control group using conventional medium. The period from June to November 2019 was analysed through available data from the history of ART Health Institution Medico S, a member of the Pronatal group. All patients underwent ET after stimulation of the cycle according to a short protocol. ETs were done on Day 2, 3, or 5 in the blastocyst stage. Age and smoking status were recorded. Statistical processing was performed using the IBM SPSS software, version 15. All obtained data were processed by standard procedures of comparative statistics, Student's t-test and Chi-squared test, with statistical significance set as p < 0.05. In the group of patients who used EG medium, the success rate of embryo implantation (positive β-hCG) on the Day 2 was 56 % (9 out of 16 patients), on the Day 3, 45.8 % (11 out of 24 patients) and on the Day 5, 30 % (3 out of 10 patients) (Figure 3).
Comparative analysis using Student t-test between these two groups did not show a statistical difference p = 0.834. However, a higher rate of embryo implantation success was observed on the second day of transfer in the group of patients using EG (Figure 4).  of 41-45, there were 66.7 % of patients with positive β-hCG in the group with EG medium (2 of 3 patients), and there were no implantation in the control group ( Figure 5).

c) Influence of patient age
Student-t test did not reveal a significant difference in age between these two groups (p = 0.182). By analysing the success rate of the implantation rate within the age groups, in the EG group a significant increase in the embryo implantation rate was observed in patients older than 35 years.

d) Impact of tobacco smoking
Impact of tobacco smoking on success in the implantation of embryos was analysed. In the group with EG medium, there were 32 % of smokers and 68 % of non-smokers, and in the control group there were 38 % smokers and 62 % non-smokers. Embryo implantation (positive β-hCG) in the group of patients who used EG medium in smokers was 43.7 %, while in non-smokers it was 47.1 % (Table 1). In the control group of female patients, 21.1 % of smokers had positive β-hCG, and in the non-smoking group, β-hCG was positive in 48.3 %. Due to the small number of samples in the observed parameters, difference is not statistically significant, but it is noticed that in smokers the implantation rate was higher if they used EG during ET.

Discussion
As already mentioned, EG contains HA and nutrients that are necessary to support embryos from transfer to the moment of implantation as well as for the process of biochemical interaction during that period. The positive effect of EG medium was proven by its introduction on the market in 2003. After that, several studies published its positive effect. In 2010, an independent Cochrane Collaboration published a meta-analysis of controlled randomised studies "ET medium adhesion components". 1 Using EG for ET, the rate of clinical pregnancies increased significantly from 41 % to 50 % compared to conventional media with low or even no HA content (meta-analysis of 13 publications covering Cochrane articles in more than 3,200 patients). Analyses did not show an increase in abortions or other unforeseen circumstances (outcomes). Cochrane authors identified a clear positive effect of high concentrations of HA in transfer media. Their report published in 2020 shows an increased pregnancy rate after using high concentrations of HA in ET media. Results from this study confirm the previous conclusions of the Cochrane report. In this study, a positive effect of EG medium was proven, since the implantation rate is higher after its use, although there is no statistically significant difference in the observed parameters.
Numerous clinics in Japan that use EG transfer medium presented different results at scientific conferences between 2010 and 2013. Meta-analyses of Japanese studies involving nearly 10,000 cycles show increased implantation and pregnancy rates from fresh and cryo transfers, after the use of EG medium. 12,13 Their results confirm the previous conclusions of the Cochrane report. HA-enriched media may increase the implantation rate 14 and clinical pregnancies in patients with previous failed implants and in the third or more attempts after cryo ET. 10,15 They also report that clinical pregnancies and implantation rates were higher in embryos after cryo ET, indicating that the viscosity of the EG medium physically protects embryos after assisted hatching. 13 Zhang et al 16 and Wu et al 17 investigated whether EG has an impact on pregnancy outcome during in vitro fertilisation compared to G2 medium. In conclusion, they cite EG as a transfer medium that can improve the rate of implantation and pregnancy in the IVF program. Based on their results, there is no statistically significant difference in the age of patients, duration of infertility, number of failed cycles, endometrial thickness, number and between embryo quality and the average number of embryos in the observed groups. The results of this study show that the level of implantation is more successful in women above 35 years of age who used EG, while in women younger than 35 the rate of implantation in both groups is approximately the same. Urman et al confirm the beneficial effect of HA-enriched medium, which is evident in women over 35 years of age, in women with poorer embryo quality and with previous unsuccessful implantation. 6 Svobodová et al 18 observed a statistically significant beneficial effect of EG medium on embryo implantation in patients aged 30-38 years. This research confirms the positive effect of EG medium on Day 2 of transfer, which shows that embryos in the early phase of transfer have a higher chance of more successful implantation with this medium, while for transfers 3 and 5 days the results are similar between the control and EG groups. Urman et al found that HA-enriched media increased the rate of clinical pregnancies and implantation on days 3 and 5 of ET. 6 Several authors state that HA-enriched media have not shown significant results in their study 2,19 and concluded that future research should show whether EG is the highest quality option for all patients or only for those patients in whom the quality of the embryo is poor, for patients in whom implantation has failed and older patients. 20 Nishihara et al 21 state that pregnancies, implantation, and abortion rates where media with different concentrations of HA was used during ET gives similar results.
Toxic components of cigarette smoke affect the gametes, ovarian, tubal, endometrial and myometrial components. Uterine receptivity is also impaired. 22 When comparison was made on the effect of EG on embryo implantation in smoking and non-smoking patients, conclusion was made that support the justified use of EG in women who smoke, because the rate of embryo implantation in women smokers who used EG as a medium is significantly higher than those who used a conventional medium. In non-smoking women there was no large difference in embryo implantation between these two media. The sample was not large enough to have a statistically significant difference.