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A tilting embryo culture system increases the number of high-grade human blastocysts with high implantation competence

Reproductive BioMedicine Online

15 March 2013

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Tetsuaki Hara, MD, PhD received his MD from the Hiroshima University School of Medicine in 1980. Following his residency and completion of his doctorate in 1991, he became a university instructor in 1991, assistant professor in 1996 and associate professor of gynaecology and obstetrics in 1999, at Hiroshima University School of Medicine. Since 2007, he has worked as a director of the division of reproductive medicine, Hiroshima Prefectural Hospital. Currently, his research interests include ovarian reserve, ovarian stimulation, polycystic ovary syndrome, single-embryo transfer, embryo culture and gynaecological endoscopic surgery.

PII: S1472-6483(12)00699-2

doi:10.1016/j.rbmo.2012.11.014

Abstract 

Human embryos normally experience mechanical stimuli during development in vivo. To apply appropriate stimuli to embryos, this study group developed a tilting embryo culture system (TECS) and investigated whether it could improve the grade of fresh human embryos compared with a control static culture system. A total of 450 retrieved oocytes from 32 IVF or intracytoplasmic sperm injection cycles of 32 women were cultured for 5–6days. Oocytes were divided randomly into TECS and control groups and then were inseminated in vitro. All embryos were evaluated at days 3 and 5 using standard grading criteria for embryo quality. The rates of fertilization per mature oocyte and high-grade cleavage-stage embryo formation in the TECS group were similar to those in the control group. The rates of blastocyst formation and of blastocysts graded 3BB or higher at day 5 were significantly higher in the TECS group than those in the control group: 45.3% (67/148) versus 32.1% (51/159) (P=0.018) and 29.1% (43/148) versus 17.6% (28/159) (P=0.018), respectively. The TECS group produced more high-grade blastocysts than the control group. Embryo movement or mechanical stimulation during embryo culture may be beneficial for human embryonic development.

A culture system that produces high-quality blastocysts capable of implantation is critically important for IVF and embryo transfer. Human embryos normally experience mechanical stimuli during development in vivo. To apply appropriate stimuli to embryos, we developed a tilting embryo culture system (TECS) by placing a culture dish on an automatically tilting plate to move embryos back and forth along the bottom of the dish. We investigated whether the TECS could improve the grade of fresh human embryos to be transferred compared with that of a control static culture system. A total of 450 retrieved oocytes from 32 IVF or intracytoplasmic sperm injection cycles of 32 women were cultured for 5days. The oocytes were divided randomly into TECS and control groups and inseminated in vitro. In the TECS group, the dishes were subjected to a maximum 20° tilt for 10min in each direction at 1° per second. All embryos were evaluated at days 3 and 5 using standard embryo quality grading criteria. The rate of fertilization and high-grade cleavage-stage embryo formation in the TECS group were similar to in the control group. The rate of blastocyst formation and growth of blastocysts graded 3BB or higher were significantly higher in the TECS group than in the control group: 45.3% (67/148) versus 32.1% (51/159) and 29.1% (43/148) versus 17.6% (28/159), respectively. The TECS produced more high-grade blastocysts than the control group, which increased the number of usable blastocysts, by exposing embryos to normal levels of mechanical stimuli in vitro.

Introduction 

The development of a reliable culture system to increase the number of usable embryos with high implantation competence in one oocyte retrieval cycle is critically important for IVF and embryo transfer. Some reports of culture systems to control chemical and mechanical microenvironments for in-vitro mammalian embryo culture have been published, such as a microwell approach (Vajta et al., 2000, Hashimoto et al., 2009, Ebner et al., 2010), pulsative mechanical microvibration (Isachenko et al., 2010, Mizobe et al., 2010) and dynamic culture systems with fluid motion (Suh et al., 2003, Cabrera et al., 2006, Smith and Takayama, 2007, Blockeel et al., 2009, Heo et al., 2010, Smith et al., 2011, Swain and Smith, 2011). For human embryo culture, microwell culture increases the cell number of the inner cell mass in blastocysts (Hashimoto et al., 2009), and pulsative mechanical microvibration improves the pregnancy rate regardless of the day of embryo transfer (Isachenko et al., 2010). These results suggest that human embryonic development in vitro can be significantly improved by optimization of chemical and mechanical microenvironments.

In vivo, human embryos are normally transported to the uterine cavity from the Fallopian tube, where fertilization and early embryogenesis occur. Phasic contraction of the smooth muscle in the wall of the Fallopian tube and the currents produced by its ciliated epithelium enable the Fallopian tube to act as a peristaltic pump to push the embryo towards the uterotubal junction (Lyons et al., 2002, Lyons et al., 2006, Zervomanolakis et al., 2007). This movement likely stimulates the embryo continuously during transport, which may also be affected by direct contact, because the tubal lumen from the ampulla to the isthmus and the diameter of an embryo are similar. Such motion and contact presumably provide mechanical stimuli, such as shear stresses, compression and frictional forces, from the tubal fluid. Mechanical stimuli can induce the proliferation and differentiation of many cell types such as endocytes, muscle cells and osteoblasts (Wang and Thampatty, 2006). Therefore, mechanical factors in the Fallopian tube might play an important role in embryonic development. However, the static culture conditions used in conventional IVF do not mimic these mechanical stimuli for embryos.

In a previous study (Matsuura et al., 2010), to apply appropriate mechanical stimuli for routine clinical use, this study developed a tilting embryo culture system (TECS) by placing a conventional culture dish on an automatically tilting plate to move embryos back and forth along the bottom of the dish using mouse embryos or donated human embryos destined to be discarded. It was found that the TECS was safe for embryos and significantly increased blastocyst cell numbers under mechanical stimuli without inducing apoptosis. The current study investigates whether the TECS increases the numbers of morphologically high-grade human blastocysts for embryo transfer or cryopreservation compared with those obtained by a control static culture system.

Materials and methods 

Patients 

This study was approved by the Institutional Review Boards of Hiroshima Prefectural Hospital (reference no. H19-10, approved 31 August 2007) and Okayama University (reference no. 420, approved 24 April 2007). After written informed consent was obtained from patients, conventional IVF or intracytoplasmic sperm injection (ICSI) cycles were carried out in 32 women at the division of reproductive medicine, Hiroshima Prefectural Hospital from March 2008 to May 2010. Patients were enrolled when 10 or more oocyte–cumulus-complexes were retrieved. The standard infertility work up was carried out for all patients and male partners.

The patient characteristics are summarized in Table 1. The exclusion criteria were women older than 40years, severe male factor disorders such as oligoasthenoteratozoospermia or azoospermia and endocrinological disorders such as a hypothalamic/pituitary disorder (World Health Organization type I; ESHRE Capri Workshop Group, 1995), hyperthyroidism, hypothyroidism or hyperprolactinaemia. Women diagnosed with polycystic ovary syndrome classified according to the Rotterdam criteria (The Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004) were included.

Ovarian stimulation 

A gonadotrophin-releasing hormone (GnRH) analogue long protocol (n=25) and GnRH antagonist flex and multidose protocol (n=7) were used for ovarian stimulation. The GnRH analogue long protocol has been described elsewhere (Hara et al., 2005). Briefly, pituitary secretion was down-regulated by administration of the GnRH analogue buserelin (Suprecur; Mochida Pharmaceutical, Tokyo, Japan) as a nasal spray. Pituitary down-regulation was confirmed by both transvaginal ultrasonography and serum oestradiol measurement. Then, injections of recombinant FSH (150–225IU/day, Follistim; Schering-Plough, Osaka, Japan) were started. In the GnRH antagonist protocol, injections of recombinant FSH (150–225IU/day) were started following menstrual cycle control with oral contraceptive pills (Planovar combination tablets; Pfizer Japan, Tokyo, Japan) for 12days. When the dominant follicle exceeded 14mm in diameter, a GnRH antagonist (0.25mg cetrorelix, Cetrotide; Shionogi, Osaka, Japan) was administered daily until ovulation was triggered. An intramuscular injection of human chorionic gonadotrophin (HCG, 5000IU; Aska Pharmaceutical, Tokyo, Japan) was administered when at least two follicles exceeded 17mm in diameter. Oocytes were retrieved at 34–36h after the HCG injection.

Randomization of retrieved oocytes 

Retrieved oocytes were randomly divided into TECS and control groups. Randomization was performed using a computer-generated list of random numbers (n=450). Oocytes were allocated to TECS or control groups if the number was even (zero was regarded as even) or odd, respectively. The TECS was applied to the allocated oocytes during preincubation before IVF or ICSI. After the oocyte was allocated and cultured for 4h in human tubal fluid (Irvine Scientific, Santa Ana, CA, USA), at 37°C with 5% CO2, 5% O2 and 90% N2, it was inseminated for 2h with spermatozoa selected previously by density gradient centrifugation and a swim-up procedure. ICSI was used for insemination when repeated analyses showed total sperm counts in the treated semen were below 1×106. ICSI was performed by standard procedures.

Tilting embryo culture system 

The TECS equipment (Strex, Osaka, Japan) was developed to apply appropriate ‘natural’ mechanical stimuli to embryos in vitro (Matsuura et al., 2010). The TECS is an electrical device with a power cord which is designed to be used in a standard humidified incubator. It consists of a control unit and a waterproof motor unit with a tilting plate (Figure 1), one-well culture dishes (IVF One Well Dish, 353653; BD Falcon, Tokyo, Japan) and/or four-well dishes (IVF MultiDish, 144444; Nunc, Roskilde, Denmark) were set on the plate. Motion parameters, such as the uniform radial velocity, maximum tilt angle and holding time at the maximum tilt angle, were set and applied by the control unit outside of the incubator. Dishes were subjected to a maximum 20° tilt in each direction at 1°/sec. This setting allowed embryos to move back and forth along the bottom of the dish at approximately 1mm/min (Matsuura et al., 2010).

Embryo culture, cryopreservation and elective single blastocyst transfer 

Fertilization was determined to have occurred when two pronuclei were identified at approximately 20h after IVF or ICSI. Fertilized oocytes continued to be cultured in sequential medium (BlastAssist system; Jyllinge, Denmark) at 37°C with 5% CO2, 5% O2 and 90% N2 for 5days in group culture without an oil overlay. The embryo density was 150μl/embryo. Three to six oocytes or embryos were cultured in a one-well culture dish with 450–900μl medium. One or two oocytes or embryos were cultured in a four-well culture dish with 150–300μl medium.

Embryos were evaluated at day 3 in terms of the number, symmetry and granularity of the blastomeres, type and percentage of fragmentation, the presence of multinucleated blastomeres and degree of compaction, as described previously (Alikani et al., 2000). High-grade day-3 embryos were characterized by having no multinucleated cells and consisting of 7–9 cells. Furthermore, in such embryos, less than 15% of the volume of the embryo should have contained fragmentation, and the embryo should have appeared symmetrical with only slightly asymmetric blastomeres. Embryos at day 5 were evaluated using Gardner’s criteria (Gardner et al., 2000) for a blastocyst. Blastocysts scoring 3BB or higher were designated as high grade. Blastocysts containing a grade C inner cell mass or trophectoderm were not designated high grade. Morulae and blastocysts scoring less than 3BB at day 5 were cultured to day 6.

The highest-grade blastocyst of each cycle, defined as the most morphologically advanced with a grade of 3BB or higher at day 5, in either group was transferred into patients at day 5 when the blastocyst was eligible for elective single blastocyst transfer according to the hospital’s policy. When the embryo grade was the same in either group, the control-cultured blastocyst was transferred. No embryos underwent assisted hatching before a fresh embryo transfer was performed. Blastocyst transfer was carried out using a Kitazato ET 3.0 Fr Catheter (Kitazato Medical, Tokyo, Japan) under transvaginal ultrasonographic guidance. Cryopreservation of supernumerary blastocysts scoring 3BB or higher at day 5 was carried out by vitrification (Kuwayama et al., 2005). All blastocysts graded 3BB or higher were vitrified if a patient was at risk of ovarian hyperstimulation syndrome or if the ovarian stimulation used the GnRH antagonist protocol, according to the hospital’s IVF/ICSI policy. When only morulae or lower-grade embryos were available, no fresh embryos were transferred. After embryos were cultured up to day 6, blastocysts scoring 3BB or higher were also cryopreserved by the same method. Conventional IVF or ICSI procedures were carried out by the same blinded embryologist. Embryos were evaluated by one embryologist who was blinded to the culture allocation, as were the clinicians, nurses and embryologists who carried out embryo transfers.

Vitrified–warmed blastocyst transfer cycles and outcomes of embryo transfer 

Micronized vaginal progesterone (450mg/day; Nacalai Tesque, Kyoto, Japan) and a transdermal oestradiol patch (Estrana tape, 0.72mg; Hisamitsu Pharmaceutical, Tokyo, Japan) were used for luteal support in women at least until a pregnancy test was carried out for a fresh blastocyst transfer. All vitrified–warmed blastocyst transfers were performed during artificial hormone replacement cycles with transdermal oestradiol patches and micronized vaginal progesterone using identical endometrial preparation protocols. Biochemical pregnancy was determined by elevation of serum βHCG over the detection range of the assay without visualization of a gestational sac by transvaginal ultrasonography. Clinical pregnancy was determined by demonstration of a gestational sac by transvaginal ultrasonography. After delivery, medical records were obtained detailing each patient’s obstetric history as reported by their obstetricians. The outcomes of embryo transfer were followed up until June 2011, when some cryopreserved blastocysts were warmed and discarded because a couple wished to discontinue storage of cryopreserved blastocysts. Serum oestradiol and βHCG were measured using an automated electrochemiluminescence immunoassay with a Cobas E601 Analyser (Roche Diagnostics, Mannheim, Germany).

Power analysis 

To detect an increase of 15% in grade 3BB or higher blastocyst formation rate (on day 5) (i.e. from 20% to 35%) for fertilized oocytes, 137 fertilized oocytes in each group would be needed to achieve a power of 80% with a chi-squared test at a significance level of 5%. This increase in grade 3BB or higher blastocyst formation rate was based on previous results at the division of reproductive medicine, Hiroshima Prefectural Hospital during 2008 (T. Hara, unpublished results).

Statistical analysis 

Continuous data were shown as the mean±SD. Categorical data were analysed using chi-squared tests to compare the rates of fertilization, cleavage, high-grade cleavage-stage embryo formation, blastocyst formation and grade 3BB or higher blastocyst formation in the two culture systems. Variables associated with embryo grade in the univariate analysis (patient’s age at oocyte retrieval (⩽34 or 35years), type of infertility (primary or secondary), cause of infertility (tubal, endometriosis, unknown or male), type of ovarian stimulation (antagonist or agonist methods), type of insemination (conventional or ICSI) and culture system (TECS or control)) were included in a multivariate analysis. A multiple logistic regression analysis with a stepwise method was used to determine the impact of grade 3BB or higher blastocyst formation. Significant differences, odds ratios (OR) and their 95% confidence intervals (95% CI) were calculated from the model’s coefficients and their standard deviations. A value of P<0.05 was considered statistically significant. JMP 7 computer software (SAS Institute) was used for statistical data analysis.

Results 

The demographic characteristics of the couples, the type of ovarian stimulation, the result of the semen analysis and the method of insemination are summarized in Table 1. Thirty-two women were enrolled in this study. The response to ovarian stimulation was good in all patients. The mean number of retrieved oocytes was 17.0±4.9 (range 10–29). Semen analysis of most of the male partners confirmed normozoospermia. However, seven cases underwent ICSI according to the hospital’s criteria for male subfertility. The clinical background and final embryonic development after TECS or control culture for each case are summarized in Supplementary Table S1 (available online only).

The embryonic development rates in the TECS and control groups are summarized in Table 2. Fertilization and cleavage rates were not significantly different between groups. The rate of formation of high-grade cleavage-stage embryos at day 3 was not significantly higher in the TECS group, compared with that in the control group. The rates of blastocyst formation at days 5 and 6 from fertilized oocytes were significantly higher in the TECS group compared with those in the control group: 45.3% (67/148) versus 32.1% (51/159) (P=0.018) and 48.0% (71/148) versus 35.2% (56/159) (P=0.023), respectively. The rates of blastocyst graded higher than 3BB at days 5 and 6 from fertilized oocytes were also significantly higher in the TECS group compared with those in the control group: 29.1% (43/148) versus 17.6% (28/159) (P=0.018) and 31.1% (46/148) versus 19.5% (31/159) (P=0.019), respectively.

Values are n/total (%). Chi-squared tests were used to compare embryonic development rates and the Mann–Whitney nonparametric U-test was used to compare the effectiveness of producing the highest-grade blastocysts between the two systems. P<0.05 was considered to be statistically significant.Control=static embryo culture system; D=day; NS=not statistically significant; TECS=tilting embryo culture system.

Among the variables associated with embryo grade, the culture system was only related to blastocysts graded higher than 3BB at day 5, and the TECS was significantly superior to static culture (P=0.018) in the univariate analysis (Table 3). In multiple logistic regression analysis, patient’s age and culture system were independent factors to predict formation of blastocysts graded higher than 3BB at day 5 (R2=0.034, model P=0.049) (Table 4). The formation of blastocysts graded higher than 3BB from patients aged 35years at day 5 was significantly inferior to that in patients aged ⩽34years (OR 0.495, 95% CI 0.257–0.935, P=0.032). The formation of blastocysts graded higher than 3BB in the TECS group at day 5 was significantly superior to that in the control group (OR 1.936, 95% CI 1.126–3.371, P=0.017).

The outcomes of embryo transfer per person are shown in Supplementary Figure 1. Among 32 patients, at least one grade 3BB or higher blastocyst up to day 6 developed using the TECS or static culture for 25 patients. Among the 25 patients that received a blastocyst from the TECS or the control group, 24 patients showed positive serum βHCG, 21 became clinically pregnant and 20 had a live birth. Twenty-two babies were born because two patients delivered twice. In seven cases, either fresh embryo transfer or cryopreservation was not performed because no grade 3BB or higher blastocysts developed using either the TECS or static culture. In 11 cases, fresh embryo transfers were performed. In 13 cases, all embryos were cryopreserved to prevent ovarian hyperstimulation syndrome or because a GnRH antagonist protocol had been used.

The outcomes of embryo transfer per embryo are shown in Supplementary Figure 2 and summarized in Table 5. Among the seven fresh embryo transfers from the TECS group, all embryos developed to a successful birth. The rest of the embryos in the TECS group (n=39) were cryopreserved for the clinical reasons described in the materials and methods. Among them, 21 vitrified blastocysts were warmed and then transferred. Of the 21 blastocysts transferred, 19 blastocysts resulted in a positive serum βHCG, 16 developed to a clinical pregnancy and 11 to a successful birth. In total, 26 blastocysts resulted in positive serum βHCG, 23 in a clinical pregnancy and 18 in a successful birth. The positive serum βHCG rate of embryos from the TECS group was significantly higher than those from the control group: 92.9% (26/29) versus 66.7% (6/9) (P=0.046).

Discussion 

As far as is known, this is the first report that quantitatively defines appropriate mechanical stimulation of human embryos in vitro to improve the production of high-grade blastocysts for implantation, pregnancy and birth rates. This study confirms that the level of mechanical stimuli applied during embryo culture is not detrimental through all embryonic stages from fertilization to the blastocyst. The retrieved oocyte–cumulus-complexes from each patient were divided randomly into either a TECS or a control group before insemination or ICSI, which decreased the selection bias. The rates of formation of blastocysts graded 3BB or higher from fertilized oocytes by the TECS method were also greater than those obtained with a conventional static culture system. Thus, the TECS approach increases the number of blastocysts and would increase the cumulative number of pregnancies or live births in elective single blastocyst transfer cycles using either fresh or cryopreserved blastocysts. Thus far, the finding that a TECS enhances the grade of embryos has not been reported.

There are two possible reasons for why the formation rate of higher-grade blastocysts was higher. First, in this study, the TECS was applied to oocytes before IVF or ICSI. Similar stimuli have been applied to embryos after fertilization by other institutions (personal communications: Yoshimasa Asada and Tetsunori Mukaida). The effect of mechanical stimuli on the fertilization of human oocytes remains unclear, although mechanical stimuli play fundamental roles in fertilization in other species (Knoll et al., 2003, Horner and Wolfner, 2008). In this study, the fertilization rate in the TECS group was not significantly higher than the control group. This result suggests that mechanical stimuli might enhance cell proliferation, rather than fertilization, via unknown mechanisms. Mizobe et al. (2010) reported that cytoplasmic maturation of in-vitro-matured pig oocytes is enhanced by mechanical vibration, whereas Isachenko et al., 2010, Isachenko et al., 2011 reported that the blastocyst formation rate of 2PN zygote increases by 10% using a mechanical vibration device. These reports suggest that appropriate mechanical stimuli before fertilization by the TECS used in this study might stimulate subsequent oocyte maturation as well as cell proliferation in the embryo. What needs to be clarified is the optimal time to apply the TECS during embryo culture, i.e. just after oocyte retrieval or after recognition of fertilization. Second, the blastocyst conversion rate was somewhat low in the control group of this study. The control conditions used in the study may be suboptimal and would explain the significant increase in blastocyst formation using the TECS over a suboptimal system, which may be another reason why this study has shown a positive improvement following TECS, although other studies using a TECS have not.

One limitation is the ending of the study before transfer of all cryopreserved–warmed blastocysts, when a couple wished to discard their cryopreserved blastocysts. The implantation rate of blastocysts from the TECS group was significantly higher than that of blastocysts from the control group. However, the clinical pregnancy and birth rates from blastocysts obtained from the TECS group were not significantly higher than those from the control group, although the rates were higher for the TECS group than those in the control group. However, considering the difference in rates between the TECS and control groups, more power would clarify the superiority of the TECS over static culture. Such cumulative data need to be calculated after the transfer of fresh blastocysts and the transfer of all cryopreserved blastocysts. For this reason, this study could not clarify that the clinical pregnancy or birth rates from blastocysts from the TECS group were significantly higher than those from the control group. The second limitation of this study was that it enrolled only good responders to ovulation induction regimens. It is still unclear whether the TECS enhances the rate of formation of high-grade blastocysts from women who are poor responders to ovulation induction regimens. To address these important questions, a multicentre prospective randomized case-control trial is currently being prepared, which will include a broader range of patients.

A culture system using microfluidic technology has been reported to enhance mouse embryonic development and pregnancy rates (Cabrera et al., 2006, Heo et al., 2010). Recently, this system has been applied to a clinical situation and reported to increase the production of high-quality human cleavage-stage embryos through a reduction of embryo fragmentation (Alegretti et al., 2011). Although this system is sophisticated and promises to improve embryonic development, the device that controls the continual pulsatile or peristaltic fluid flow is very specific and quite complex to use. In contrast, the TECS can be rapidly implemented in a laboratory, because it can be fitted to a standard incubator and adapted to different types of culture dishes. Using conventional culture dishes in this study, the formation rate of blastocysts graded 3BB or higher was significantly greater using the TECS compared with the conventional static culture system.

Excessive shear stress has been reported to cause physical damage to embryos (Xie et al., 2006, Xie et al., 2007). Thus, shear stresses exceeding 1.2dynes/cm2 result in death of the blastocyst within 12h (Xie et al., 2006). However, a previous study showed that the velocity of embryo movement on the TECS plate is approximately 0.1mm/min, equivalent to shear stress of only 0.007dynes/cm2 (Matsuura et al., 2010). Moreover, the shear stresses produced by the TECS are not harmful to mouse or human embryos destined to be discarded (Matsuura et al., 2010). The previous study also demonstrated that the cell numbers of mouse and human blastocysts cultured using the TECS are greater than those of blastocysts cultured in a static culture system, suggesting that the TECS enhances the rate of cell division in embryos. According to other reports (Xie et al., 2006, Cui et al., 2008), the number of cells in mouse embryos is negatively correlated with the percentage of apoptotic cells. In bone and endothelial cells, downstream transcription factors in the nucleus are activated by mechanical stimuli such as shear stresses, and mechanotransduction, gene transcription and DNA synthesis are also activated (Wang and Thampatty, 2006). Cell numbers might increase in the absence of apoptosis as a result of the enhancement of cell division induced by these activations.

Another explanation for the enhancement of cell division is facilitated diffusion of waste products from cultured embryos in the TECS. After addition of a microsphere to the centre of a 50μl microdrop, the times for the microsphere to reach the edge of the microdrop were 60 and 5min under a static condition and using the TECS, respectively. Facilitated diffusion reduces the autocrine effect in the TECS, because the concentration of products for embryo growth near the embryo decreases faster compared with that in static culture, which is consistent with microfunnel results (Heo et al., 2010). Under the culture conditions of this study, embryo numbers in the culture medium were between one and six, and the embryo concentration was 150μl/embryo. Decreasing the culture medium volume per embryo may result in a beneficial effect using the TECS. Group culture of human embryos in vitro improves their development, probably due to paracrine and enhanced autocrine factors by the paracrine interaction (O’Neill, 2008, Ebner et al., 2010). Paracrine mediators would not be concentrated by the facilitated diffusion with fluid motion, and paracrine factors would not be dominant for the improvement of human embryonic development using the TECS. At present, it cannot be determined which mechanical stimuli or/and diffusion of waste products in the culture medium is dominant for the improvement of embryonic development. Delineating the molecular responses of human embryos, such as with intracellular calcium ion and 1,4,5-triphosphate concentrations, to mechanical stimuli may help to clarify the enhancement of cell division using dynamic culture systems (Isachenko et al., 2010).

In conclusion, a tilting embryo culture system was developed to apply appropriate mechanical stimuli such as shear stress to embryos in vitro for clinical assisted reproduction, which leads to a higher rate of production of high-grade blastocysts with a higher implantation potential than those obtained from a static culture method. The system is a promising culture method that enhances the numbers of usable blastocysts in a single oocyte retrieval cycle by exposing them to mechanical stimuli similar to those found in the Fallopian tube.

Acknowledgements 

This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas to KN (‘System cell engineering by multi-scale manipulation’, no. 17076006) and Special Coordination Funds for Promoting Sciences and Technology to KM from the Japanese Ministry of Education, Science, Sports and Culture.

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Date Added: 15 March 2013   Date Updated: 15 March 2013
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