Improvement of the embryological stage in assisted reproduction treatment with hyaluronan-enriched culture medium in couples using preimplantation genetic testing for aneuploidy

The studies on the improvement of the effectiveness of infertility treatment using assisted reproductive technologies (ART) are currently directed at the development of new modified schemes and medications to stimulate the ovarian function, as well as the improvement of the embryological stage, in particular the conditions of embryo cultivation [1]. According to the studies, implantation failure of the embryo of good/excellent quality can result from the inadequate formation of the embryo-endometrial adhesion [2]. Hyaluronic acid contributes to the formation of a stronger connective base between the embryo and endometrial cells. The acid acts through CD44 receptors which are expressed on the surface of the embryo at the cleavage stage and in the endometrium during the implantation window [3, 4]. To date, there is a large number of commercial single-component and multi-component culture media. However, despite all the achievements in embryo cultivation, including the development of various culture media, it is worth considering the difference between the natural environment for embryo development and in vitro conditions. Hyaluronan-enriched culture media imitate the natural environment in the uterine cavity during implantation [5]. The high concentration of hyaluronic acid makes the culture medium thicker and minimizes embryo drift. Hyaluronan-enriched culture media contain purified or recombinant albumin, which serves as an energy source for the developing embryo and also regulates osmotic pressure [6]. Moreover, albumin addition to the culture medium increases its viscosity and prevents the embryo from attaching to the culture plastic or catheter [7].

At the moment, there are contradictory data on the effectiveness of the culture medium enriched with hyaluronic acid and recombinant albumin in relation to the pregnancy rate and live birth rate in patients undergoing infertility treatment using ART. The results of one of the first studies on the use of culture media with hyaluronic acid showed an increase in the pregnancy rate in patients with repeated ART failures and a history of recurrent miscarriage [8]. The Cochrane review published in 2014 described an increase in pregnancy rate by 8% in patients using ART with the addition of hyaluronic acid to the culture medium of the embryo [9]. The subsequent analysis, which included the data of 3898 patients, demonstrated an increase in live birth rate when the embryo was cultivated in the medium with hyaluronic acid; however, an increase in the multiple pregnancy rate was noted. Another study conducted in China in 2011 showed a decrease in the ectopic pregnancy rate when culture medium with a high content of hyaluronic acid was used [10]. However, Fancsovits et al. (2015) questioned the available data on the positive effect of hyaluronic acid on embryo implantation after studying the results of 581 cases of embryo transfer in IVF/ICSI cycles. The researchers showed the absence of the influence of hyaluronan-enriched culture medium on the implantation rate [11]. The routine use of the culture medium with high concentration of hyaluronic acid was not recommended by S. Safari et al. either, since the results of their study, which included 229 patients, did not show a statistically significant increase in the implantation rate as well as the rate of pregnancy and delivery when hyaluronic acid was used [12]. Similar data on the absence of the effect of hyaluronic acid on embryo attachment to the endometrium were obtained by Ruane et al. (2020) in an in vitro experiment [13]. However, the authors suggested that, on the one hand, in vitro conditions cannot be fully identical to the conditions under which the implantation of a blastocyst occurs in a woman’s body, and on the other hand, the effect of culture medium with hyaluronic acid on the increase in the pregnancy rate and further development of pregnancy may be associated with other mechanisms rather than a tighter attachment of the embryo to the endometrium.

Another reason why patients cannot achieve pregnancy is the impairment of the chromosomal status of the embryo during the transfer of a good/excellent quality blastocyst [14]. Preimplantation genetic testing for aneuploidy (PGT-A) makes it possible to identify euploid embryos at the preimplantation stage, which leads to an increase in pregnancy rate, decrease in reproductive losses and decrease in the risk of having children with genetic disorders. Therefore, the transfer of the euploid embryo in the subsequent cryopreservation protocols made it possible to minimize the influence of the embryonic factor on the interpretation of the obtained data and to assess the real effect of the culture medium with hyaluronic acid and recombinant albumin on the rate of pregnancy and delivery.

The aim of this study was to analyze the implantation rate, pregnancy and live birth rates in patients undergoing infertility treatment with IVF/ICSI with PGT-A when an euploid embryo is transferred in hyaluronan-enriched culture medium.

Materials and methods

This was a retrospective analysis of the data of 309 couples who underwent IVF/ICSI cycles and PGT-A with the subsequent transfer of the euploid embryo; the study was conducted at the B.V. Leonov Department of Assistive Technologies in the Treatment of Infertility. The study was approved by the Local Ethics Committee of the National Medical Research Centre for Obstetrics, Gynecology and Perinatology, Moscow, Russia.

All couples who were included in the study underwent a preliminary examination in accordance with the Order of the Ministry of Health of the Russian Federation No. 803n and signed an informed consent. IVF/ICSI cycles with gonadotropin-releasing hormone (GnRH) antagonist started on the 2nd–3rd day of the menstrual cycle of the patients. When the follicles reached a diameter of 18 mm, a trigger for the final maturation of oocytes (hCG) was administered to the patients. To prevent ovarian hyperstimulation syndrome, the trigger was replaced with GnRH agonist in 100 patients. Given the depletion of the ovarian reserve, natural IVF/ICSI cycles were carried out in 5 patients. Patients who used donor oocytes or donor sperm, as well as patients over 44 years were excluded from the study. The average age of women included in the study was 34 years, the average age of men was 38 years. All couples underwent next-generation high-throughput sequencing (NGS) for PGT-A testing of good/excellent quality embryos. If euploid embryo was not obtained after PGT-A, such patients were excluded from the study.

After transvaginal puncture of the ovaries, fertilization of the oocytes was carried out by ICSI; then the fertilized cells were transferred to CSCM medium (Irvine Sc., USA) for further cultivation. The stage of two pronuclei (zygote formation) was evaluated 14–16 hours after fertilization. All stages of cultivation were carried out in a multi-gas incubator (COOK, Ireland) in drops of 25 µl under oil (Irvine Sc., USA). CSCM medium (Irvine Sc., USA) was not changed during 5 days of cultivation. On the 5th day after fertilization, trophectoderm biopsy was carried out which was followed by cryopreservation of biopsied embryos. Cryopreservation and subsequent defrosting of the embryo in a cryopreservation protocol were carried out using vitrification kits Kitazato (Japan), Irvine (USA), Z-VIT (Russia) and Z-REV (Russia) according to the manufacturer’s recommendations. PGT-A consisted of several stages: whole genome amplification and library preparation for applying it to the chip were carried out at the first stage. Special molecular tags, namely barcodes, which are unique for each sample, were attached to the DNA fragments to create a library. Then ionic semiconductor sequencing was performed; it was followed by bioinformatic analysis of the results and the conclusion based on the data obtained according to the standard PGT-A procedure.

After receiving the results of PGT-A, the endometrium was prepared with estrogen-containing drugs starting from the 5th-6th day of the menstrual cycle of a patient. Embryo transfer medium with increased viscosity was used in group 1. This medium contained hyaluronic acid in high concentration, saccharides and amino acids, as well as recombinant albumin as a protein source (53 couples). Embryo transfer was carried out according to the manufacturer’s instructions. Cryotransfer of embryos in patients of the control group was carried out according to the standard scheme using a medium that did not contain hyaluronic acid (256 couples).

The day before embryo transfer, 2.0 ml hyaluronan-enriched culture medium was placed under oil into the dish and left for 15 hours at a temperature of 37°C and 6% CO2. On the day of the transfer, the embryo was thawed according to the standard procedure. After defrosting, the embryo was immediately placed for 10 minutes in hyaluronan-enriched culture medium without precultivation. After 10 minutes, the culture medium enriched with hyaluronic acid and containing an embryo was withdrawn into a soft catheter Wallace (Germany) or CooK (Australia) in a volume of 30 µL. In cases when the culture medium was used without hyaluronic acid, the embryo transfer was carried out according to the standard embryological technique. All patients included in the study underwent selective transfer of one embryo. In both groups of patients, the embryo was transferred into the uterine cavity on the 18th-21st day of the menstrual cycle when the thickness of the endometrium was more than 8.2 mm on the day of embryo transfer. The luteal phase of the cycle as well as post transfer period were supported according to the standard procedure [15]. On the 14th day after embryo transfer, the patients had their blood tested for the beta subunit of human chorionic gonadotropin (β-hCG) to diagnose pregnancy. If β-hCG was positive, transvaginal ultrasound examination was performed to diagnose clinical intrauterine pregnancy on the 21st day after embryo transfer. In the presence of a gestational sac in the uterine cavity, repeated ultrasound assessment of the pelvic organs was performed at 7-8 weeks’ gestation. Further monitoring of pregnancy as well as the subsequent collection of the data on pregnancy outcomes was carried out by an obstetrician-gynecologist at the local antenatal clinic.

Statistical analysis

The analysis of the results was carried out using IBM SPSS Statistics software package version 23.0 (USA) and Microsoft Excel tables. The type of data distribution was determined for the analysis of quantitative data in groups (Kolmogorov–Smirnov test). Absolute numbers (N) and percentages of the total number of patients in group (P) in the N (P%) format were used to describe categorical binary data. The statistical significance of the differences between two or more indicators (frequencies, shares) was assessed using the Pearson’s chi-squared test. A non-normally distributed parameters were described as medians (Me) and quartiles (Q1 and Q3) in the Me (Q1; Q3) format. Thresholds for the p-value statistical significance was 0.05.

Results

All patients included in this study were divided into two groups: patients whose embryo was transferred in a culture medium with hyaluronic acid (group 1, n=53), and patients who had a transfer with continuous single culture product CSCM (group 2, n=256). The patients of each group were divided into subgroups depending on the implantation result. The total results of the ART program of the patients included in the study are presented in Table 1. The relative rate of clinical pregnancy in the control group was 43.7% and it was 32.0% in the comparison group. Missed abortion was revealed in 13 patients of the control group (13.1%), one patient was diagnosed with ectopic pregnancy. In the comparison group, one patient was diagnosed with missed abortion (5.8%). In the control group 15 patients had a live birth after embryo transfer in hyaluronan-enriched culture medium (88.3%) and live birth was noted in 90 women of the comparison group (80.3%) (Table 1). Eight patients of the control group were diagnosed with miscarriage in early pregnancy which was accompanied by bloody discharge and abdominal pain. The most likely cause of early miscarriage was obstetric syndromes, including disorders of the blood coagulation system.

In order to minimize the influence of age on the outcomes of ART programs and the interpretation of the results, all the patients were divided into three subgroups depending on their age: subgroup 1 (24–30 years, 72 women), subgroup 2 (31–36 years, 148 women), subgroup 3 (37–43 years, 89 women). The effectiveness of the ART treatment was analyzed in each group.

In subgroup 1, the rate of clinical pregnancy in patients who underwent embryo transfer in hyaluronan-enriched culture medium was 27.2%, and it was 42.6% in the group of embryo transfer in CSCM. All patients who had embryo transfer in hyaluronan-enriched culture medium had live births. The delivery rate was 76.9% in the control subgroup (Table 2). Five patients in this subgroup were diagnosed with early miscarriage, one patient was diagnosed with ectopic pregnancy followed by tubectomy (embryo transfer in CSCM).

The rate of clinical pregnancy in subgroup 2 was 25.0% and it was 46.8% in the control subgroup. One patient in the control group was diagnosed with anembryonia. One patient had an early pregnancy termination. The rate of live birth after embryo transfer in the culture medium with hyaluronic acid was 100% and it was 96.6% in the subgroup with CSCM (Table 3).

Embryo was transferred in a culture medium with hyaluronic acid in 22 patients of subgroup 3. Two patients in the control subgroup were diagnosed with early miscarriage and one patient became pregnant after embryo transfer in hyaluronan-enriched culture medium. Missed abortion was diagnosed in 12 patients at 7–8 weeks’ gestation (embryo transfer in CSCM). The rate of clinical pregnancy in the comparison subgroup was 40.9% and it was 38.8% in the control subgroup. The relative rate of delivery in the subgroup of patients with embryo transfer in hyaluronan-enriched culture medium was 88% and it was 50% in the control subgroup (Table 4).

The results of our study showed that there were no differences in the rate of pregnancy and delivery in subgroups of women of different age depending on the use of hyaluronan-enriched culture medium (p≥0.05). Clinical and anamnestic characteristics were analyzed within each age subgroup of patients who had embryo transfer in hyaluronan-enriched culture medium and a standard one. According to the results of the analysis, the subgroups were comparable in clinical and anamnestic data. It is worth noting that despite the absence of statistically significant results on the effect of the culture medium with hyaluronic acid on pregnancy rate and pregnancy development, there is a positive influence of such medium on the course of pregnancy which occurred after the transfer of an euploid embryo, especially in patients of older reproductive age. The absence of statistically significant results may be due to a small sample of the patients in the group of hyaluronic acid.

Discussion

Embryo implantation into the endometrium is a multi-stage process which is due to multilevel regulation of intracellular and intercellular interactions [16]. These processes are necessary for the further development of the blastocyst and the adaptation of the mother’s body to pregnancy. The effectiveness of ART programs is certainly dependent on the combination of many factors; however, successful implantation and further development of normal pregnancy are eventually due to the correct and coordinated interaction of a genetically healthy embryo with the receptive endometrium [17]. This study is unique because the embryonic factor which significantly affects the outcome of the ART treatment was minimized during the data analysis. Euploid embryos were obtained from all patients included in this study. The embryos were transferred in a subsequent cryopreservation protocol after the patient’s endometrium was prepared with cyclic hormone therapy.

A large number of papers with contradictory results have been published regarding the effect of hyaluronic acid on the pregnancy rate and pregnancy development. Under normal conditions, the uterus and fallopian tubes contain hyaluronic acid, and during implantation the concentration of hyaluronic acid increases. Hyaluronic acid binds to CD44 receptors on the surface of the endometrial epithelium and thereby regulates the expression of a number of genes involved in embryo implantation, as well as participates in the processes of cell adhesion, proliferation, migration and cellular differentiation [3, 4]. The participation of hyaluronic acid in the processes of embryo implantation under normal conditions accounts for the growing interest of scientists in the use of a culture medium enriched with this component in assisted reproduction.

We carried out the analysis of clinical and anamnestic characteristics, infertility factor, implantation rate, rate of pregnancy and delivery of all patients included in the present study. The results of the study did not show a statistically significant advantage of the culture medium containing hyaluronic acid on the outcomes of the ART program and the rate of delivery. However, one should not ignore the tendency to increase the rate of ongoing pregnancy in patients of different age subgroups, especially in patients of the older age subgroup. In subgroup 1 (24–30 years), miscarriage rate was 0% in the comparison group and 20% in the control group; in subgroup 2 (31–36 years) this indicator was 0% and 5%, respectively, and in the subgroup of patients of older reproductive age (37–43 years), miscarriage rate was 12% in women with embryo transfer in hyaluronan-enriched culture medium and 50% in patients of the control group.

In 2021, Tope Adeniyi et al. published a review of studies on the use of hyaluronic acid for embryo cultivation in ART programs. In addition, the authors analyzed the rate of pregnancy and delivery in patients depending on the embryo cultivation method [18]. The study included 1018 patients who underwent embryo transfer in culture medium with a low content of hyaluronic acid and 1175 patients who underwent embryo transfer in culture medium with a high content of hyaluronic acid. In a group of patients who underwent embryo transfer in culture medium with a high content of hyaluronic acid, the embryo was in this medium for 2–4 hours or for 10-30 minutes. The results of the study showed that the use of a culture medium enriched with hyaluronic acid statistically significantly increases the rate of pregnancy and delivery, regardless of the time of embryo cultivation in the culture medium (prolonged cultivation: OR 1.21, 95%, CI 0.99–1.48; rapid cultivation: OR 1.32, 95% CI 1.02–1.72; total OR 1.26, 95% CI 1.03–1.54 compared to the culture medium with a low content of hyaluronic acid). The results of the study showed that the duration of embryo cultivation in hyaluronan-enriched culture medium does not affect the onset of pregnancy, however, enriching the culture medium with hyaluronic acid increases the effectiveness of the ART program.

The potential positive effect of hyaluronic acid on the development of pregnancy is due to the effect of this component on the development of the embryo, as well as on the processes of decidualization of the endometrium. Moreover, hyaluronic acid can act as a source of embryonic growth factor, since small concentrations of hyaluronic acid can penetrate through zona pellucida [19].

Contradictory results of other studies may be related to the fact that a small volume of culture medium (25–75 µl) was used in different studies, whereas K. Nakagawa et al. showed that the use of 1–2 ml of the culture medium with hyaluronic acid had a positive effect on the pregnancy rate [20]. In addition, the concentration of hyaluronic acid also differs in the culture media of different manufacturers, which may affect the interpretation of study results [9]. A Cochrane review published in 2014 described the positive effect of culture media containing 0.5 mg/ml of hyaluronic acid on the pregnancy rate [9].

The results of this work confirm the necessity of conducting further studies to improve the use of culture medium enriched with hyaluronic acid, as well as to determine the indications for its use in different groups of patients. According to reviews, culture medium enriched with hyaluronic acid is most often used in patients of older reproductive age [20]. It is worth noting that in this study, the most apparent trend towards an increase in the rate of pregnancy and delivery was noticed in this age subgroup (p=0.8 and p=0.07).

Conclusion

Implantation failure of the euploid embryo can be caused by insufficient receptivity of the endometrium, as well as by inadequate formation of the blastocyst-endometrial adhesion. According to the results of previous studies, hyaluronic acid reduces the migration of the embryo in the uterine cavity and also contributes to the contact of the embryo with the endometrium. The results of numerous studies recommend the use of hyaluronan-enriched culture medium in patients of older reproductive age and patients with the history of repeated IVF failures; however, further studies with a large sample of patients should be carried out to improve and normalize the conditions of embryo cultivation in these culture media.