The first Russian experience with controlled mechanical microvibration in growing human embryos in assisted reproductive technology programs

In recent years, special attention has been paid to the selection of optimal parameters for culturing human embryos in assisted reproductive technology (IVF – in vitro fertilization) programs [1–4]. In vitro embryo development is constantly exposed to stressful effects that it would not have experienced in vivo. These include pH and temperature fluctuations, the effect of atmospheric oxygen, natural and artificial light [5]. To improve human embryo culture systems, the optimal composition of the culture medium should be chosen [6–8]. However, most systems consist of relatively small (up to 1 ml) and completely static volume of the culture medium [9–11]. Obviously, these conditions are far from optimal (in vivo) conditions [12].

In vivo fertilization and preimplantation development of the embryo occur in the fallopian tube. At this time, the embryo is in constant motion due to peristaltic contractions of the muscular wall in the fallopian tube and action of the fallopian tube villi. Muscle contractions are necessary for enveloping gametes and, subsequently, the embryo with the secretion of the fallopian tubes, which contributes to the normal fertilization process and the movement of the embryo towards the uterus [13].

The mucous membrane of the fallopian tube consists of villous epithelial cells. Their villi constantly fluctuate with a frequency from 4.9 ± 0.2 Hz in the proliferative phase to 5.8 ± 0.3 Hz in the secretory phase of the menstrual cycle [14, 15]. According to Isachenko et al. [16], the embryo is normally exposed to vibration with a frequency of up to 20 Hz. Ciliary contractions cause fluctuations in the secretion of the fallopian tube leading to the improvement of the nutrient diffusion and having a direct mechanical effect on the embryo. This results in the activation of various intracellular pathways [17] and is an important factor of the regulation of embryo preimplantation development [18].

Thus, during the preimplantation development, the embryo is in a constant and very complex dynamic interaction with its micro and macro environment. The muscular contractions of the uterus and fallopian tubes with the constant movements of the fallopian villi create unique conditions for the fertilization and subsequent embryo development [19, 20]. There is no doubt about the influence of a dynamic microenvironment on embryo development in vivo and in vitro [12].

A combination of the cultivation systems with microvibration [16, 21, 22] can be a new approach to improving the culturing conditions of human embryos in IVF programs. The aim of the study was to evaluate the effect of controlled mechanical microvibration of human embryos on the pregnancy rate in IVF programs.

Materials and Methods

A prospective study included 375 couples without contraindications or complications developed during assisted reproductive technologies programs. All patients were examined according to Order of the Ministry of Health of the Russian Federation No. 107n [23]. Among 375 patients, 75 patients were included in the microvibration group and 300 patients were included in the control group.

The non-inclusion criteria were the impossibility of embryo transfer due to preimplantation genetic testing, the use of donor oocytes, the patient’s age over 45 years, planned cryopreservation of all received oocytes or embryos. The exclusion criteria were receiving less than two MII oocytes, emergency cryopreservation of all received oocytes or embryos, canceling of the embryo transfer due to the risk of ovarian hyperstimulation syndrome, endometrial pathology, acute somatic or infectious disease, as well as patient’s desire to stop participating in the investigation. All patients signed a voluntary informed consent to participate in the research. The study was approved by the local institutional review board of the Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of the Russian Federation.

Ovarian stimulation was performed with gonadotropin-releasing hormone antagonists. Human chorionic gonadotropin or gonadotropin-releasing hormone agonist was used as an ovulation trigger. One-stage cultivation of embryos was performed. A morphological assessment of embryos was conducted by an embryologist according to Gardner’s classification (Gardner D.K., 1999) in 120–122 hours after fertilization. The embryo transfer was performed on the fifth day. Pregnancy was assessed by the β-hCG blood level in 14 days after the embryo transfer.

In the microvibration group, the incubator was placed on the ArisTT180-s platform (K&S Advanced Systems Ltd, Israel) in the active vibration mode with a frequency of 40 Hz for 30 seconds with a rest interval of 30 minutes. Culturing under the microvibration condition was carried out throughout the entire period from receiving oocytes to the embryo transfer (or cryopreservation of the embryo). The built-in oscilloscope was used to estimate the real vibration frequency and amplitude.

The GraphPad Prism statistical software package (GraphPad Software, USA) was used for statistical analysis and plotting. The generalized D’Agostino-Pearson test was used to determine the normality of the distribution. The median and interquartile range were calculated for nonparametric quantitative data, and the Mann-Whitney test was used. The absolute value and percentage were calculated for qualitative data, the exact Fisher test and the Cochran-Armitage test were used. Differences were considered statistically significant at p <0.05.

Results

There was no difference in patients’ age, body mass index (BMI), clinical data and medical history. The women’s age in the microvibration group was 33 (30–39) years, in the control group it was 34 (30–38) years, p = 0.77; the men’s age was 35 (31–38) years in the microvibration group and 34 (32–38) years in the control group, p = 0.99. BMI was 22.3 (20.9–25.6) versus 21.8 (20.1–24.3) kg/m2, respectively, p = 0.36. For 39 (52.0%) patients in the microvibration group and 159 (53.0%) patients in the control group, this IVF attempt was the first one, p = 0.49. Patients did not differ in the total number of ovarian stimulation and embryo transfer history, which ranged from 0 to 9 and averaged 0 (0–1) for both groups, p = 0.77. The average duration of infertility did not differ in the groups of comparison either and it was 5 (3–9) and 5 (3–7) years, respectively, p = 0.19.

Primary infertility was noted in the history of 39 (52.0%) patients in the microvibration group and 157 (52.3%) patients in the control group, p = 0.53. Among patients with secondary infertility, there were two pregnancies in the history in the microvibration group (1-2), and one pregnancy in the control group (1-2), p = 0.31. The average number of births, abortions, ectopic pregnancies and missed abortions was equal in both groups and for each of the pregnancy outcomes was 0 (0–1), p = 0.68, 0.77, 0.77 and 0.66, respectively.

In the microvibration group, one embryo was transferred in 52 (69.3%) patients, two embryos were transferred in 11 (14.7%) patients. Embryo transfer was canceled in 12 (16.0%) patients. In the control group, one embryo was transferred in 206 (68.7%) patients, p = 0.52; two embryos were transferred in 42 (14.0%) patients, p = 0.50. Embryo transfer was canceled in 52 (17.3%) patients, p = 0.47.

The pregnancy rate per ovarian stimulation cycle was 42.7% (n = 32) in the microvibration group versus 33.7% (n = 101) in the control group, p = 0.09. The pregnancy rate per embryo transfer was 50.8% in the microvibration group versus 40% in the control group, p = 0.097. When one embryo was transferred, the pregnancy rate was 52.0% (n = 27) in the microvibration group and 41.3% (n = 85) in the control group, p = 0.11. When two embryos were transferred, the pregnancy rate was 45.5% (n = 5) in the microvibration group and 38.1% (n = 16) in the control group, p = 0.46.

We also analyzed the pregnancy rate depending on the number of transferred embryos. The pregnancy rate when transferring one embryo was 43.4%, when transferring two embryos – 39.6% (p = 0.36).

When one embryo was transferred, the quality of the internal cell mass (ICM) and trophectoderm (TE) of the embryo was the same in the groups of comparison (Table). When two embryos were transferred, the degree of embryo maturity in the microvibration group was higher than in the control group (p = 0.02). The quality of ICM was also slightly higher in the microvibration group (p = 0.08), while the quality of TE did not differ (p = 0.16).

In the microvibration group 45 (60%) patients had embryos suitable for cryopreservation; in the control group – 165 (55%) patients, p = 0.258. The average number of cryopreserved embryos was slightly higher in the microvibration group – 3 (1.5–5) versus 2 (1-3) in the control group, p = 0.06.

Discussion

In the study, we compared the outcomes of IVF programs when culturing human embryos under standard conditions and when culturing embryos under the controlled mechanical microvibration conditions. The pregnancy rate was analyzed in 375 couples without contraindications to IVF and complications during it. Patients did not differ in age, BMI, and clinical data and medical history. Groups were also comparable in the number of transferred embryos and the frequency of embryo transfer cancellation.

According to our data, the pregnancy rate per ovarian stimulation cycle was 9% higher when embryo was cultured under the microvibration conditions, the pregnancy rate per embryo transfer was 10.8% higher. Obtained data are consistent with the data of other authors who studied the application of this technique to increase the effectiveness of IVF programs. According to Hur Y.S. et al. (2016), controlled mechanical microvibration makes it possible to increase the pregnancy rate by 5.2% [24]. According to Isachenko V. et al. (2017), it can be increased by 6-9% (depending on the patients age) [25].

The pregnancy rate did not differ depending on the number of transferred embryos, which can be partially explained by the lower quality of embryos in non-selective transfer [26, 27]. At the same time, the efficiency of microvibration was slightly higher in selective embryo transfer, although we were not able to achieve the statistical significance of the differences due to a decrease of sample size in the separate analysis of selective and non-selective transfers.

When transferring two embryos, the embryo maturity and ICM quality of the transferred embryos were higher in the microvibration group. Moreover, when transferring one embryo, these differences were not observed. This can be explained by the more valuable effect of mechanical microvibration on low and medium quality embryos. However, further studies with a different design of scientific work are needed to confirm this hypothesis [12, 24, 25, 28]. In addition, an increase in the average number of embryos suitable for cryopreservation from 2 (1-3) to 3 (1.5-5) shows the positive influence of controlled mechanical microvibration.

Conclusion

Cultivation of human embryos under controlled mechanical microvibration can be used to increase the pregnancy rate in IVF programs. Nevertheless, further studies are needed to evaluate the frequency of birth of a living healthy child and long-term outcomes to introduce this method of embryo culturing into wide clinical practice.