A method to improve embryo development potential when fertilization is delayed in mice

ABSTRACT

The purpose of this study was to explore the application of glucose to improve embryo development potential when fertilization is delayed in mice. After recovery, mouse oocytes were cultured alone for 6 h before fertilization in three fertilization media: G-IVF PLUS, G-IVF PLUS with 5 mM and 10 mM glucose. G-IVF PLUS group was used as the control group. Then, in vitro fertilization (IVF) was performed and blastocysts were transferred at Day 4. To compare the effects of different glucose concentrations on embryo development and birth outcomes, conventional IVF and embryo transfer were carried out in G-IVF PLUS, G-IVF PLUS with 5 mM and 10 mM glucose. The results indicated that G-IVF PLUS with 5 mM glucose significantly increased blastocyst rate (p < 0.05) and birth rate (p < 0.05) when fertilization was delayed 6 h compared with G-IVF PLUS groups. In conventional IVF without delayed fertilization, embryo development was not significantly affected by G-IVF PLUS with 5 mM or 10 mM glucose. There were no significant differences in terms of birth rate, fetal weight, crown-rump length, tail length and birth defect rate among the three groups. In conclusion, 5 mM glucose could significantly improve embryo developmental potential and birth outcomes when fertilization was delayed 6 h and did not have adverse effects on embryo quality and birth outcomes for normal IVF. It might have a good prospect of clinical application in assisted reproductive technology (ART).

Abbreviations: ART: assisted reproductive technology; IVF: in vitro fertilization; ICSI: intracytoplasmic sperm injection; TFF: total fertilization failure; TESA: testicular sperm aspiration.

KEYWORDS:

• Delayed fertilization
• oocyte aging
• art
• embryo development potential

Introduction

Since the first child born as a result of in vitro fertilization (IVF, Steptoe and Edwards 1978), great advances have been achieved in assisted reproductive technology (ART). However, there are still some problems to be solved. For instance, fertilization is often inevitably delayed in some situations. When sperm are not available from an ejaculate, testicular sperm aspiration (TESA) or oocyte cryopreservation may be performed. It will be a few hours before consent is obtained and the operation completed. Thus, oocytes will not be fertilized at the most optimum time. It is a challenge to find a method to improve embryo development potential when fertilization is delayed.

If fertilization does not happen for a prolonged period after ovulation, mammalian oocytes will undergo time-dependent deterioration in quality, called ‘postovulatory aging’ (Wang et al. 2017). It has been known that oocyte aging induces a series of structural changes, including cortical granule exocytosis (Díaz and Esponda 2004a), zona pellucida (ZP) hardening (Díaz and Esponda 2004b), along with increased chromosome and spindle anomalies (Saito et al. 1993). Eventually, postovulatory aging significantly compromises embryo development (Chian et al. 1992; Gao et al. 2016) and affects offspring health (Tarín et al. 1999, 2002). If we can find an anti-aging substance, we will improve developmental potential when fertilization is delayed in ART.

There are some substances that can delay oocyte aging and relive deterioration in oocyte quality, such as quercetin (Wang et al. 2017), melatonin (Nie et al. 2018) and glucose (Li et al. 2011). They can reduce aging-induced morphological changes, alleviate reactive oxygen species accumulation, and attenuate the aging-associated abnormalities in spindle organization and mitochondrial distribution. Among these, glucose is one of the conventional components of culture medium (Chronopoulou and Harper 2015) and has no cytotoxicity. Considering safety, it is most close to clinical application in human ART compared with other anti-aging substances. However, the effects of glucose on embryo development potential have not been studied when fertilization is delayed. Therefore, we supplemented the fertilization medium with glucose and compared the effects of different glucose concentrations on embryo quality after delayed and normal fertilization in mice. Furthermore, we studied the effects of glucose supplement on birth outcomes. It will help explore the application of glucose supplementation to improve embryo development potential when fertilization is delayed in human ART.

Results and discussion

The aim of the present study was to explore the application of glucose addition to improve embryo developmental potential when fertilization was delayed. We found that fertilization medium with 5 mM glucose may have clinical value.

The design of the present study is based on the results of Li et al. (2011). They found that oocytes could use glucose to prevent aging in the presence of cumulus cells (Li et al. 2011). In the process of IVF, COCs but not denuded oocytes are co-incubated with sperm. Accordingly, we chose glucose to prevent oocyte aging. But they did not define the reference glucose concentration (5.56 mM). What is more, they used a model of oocyte activation. For clinical application, we must test more glucose concentrations and demonstrate the validity and reliability of glucose on IVF embryos. In the present study, we first analyzed the effects of fertilization media with different glucose concentrations on embryo development potential when fertilization was delayed 6 h as shown in Table 1. G-IVF PLUS was used as control and the concentration of glucose was 2.58 mM before glucose addition. Glucose was added to G-IVF PLUS to make total glucose concentration up to 5 mM and 10 mM after addition. COCs were cultured alone without sperm for 6 h in control, G-IVF PLUS with 5 mM and 10 mM glucose groups which were assigned 6 h prior to fertilization. In other words, fertilization was delayed 6 h. Then, IVF was performed in the three groups and embryos were cultured to blastocysts. We found that there were no significant differences in terms of cleavage, 2-cell or 8-cell rates among the three groups. G-IVF PLUS with 5 mM glucose significantly increased blastocyst rate compared with control or G-IVF PLUS with 10 mM glucose group (p < 0.05). The number of blastocysts in Table 1 includes the number of blastocysts transferred in Table 2. The results indicate that 5 mM glucose can improve embryo development potential when fertilization is delayed 6 h. However, 10 mM glucose does not improve embryo development at the same condition.

Table 1. The effects of fertilization media with different glucose concentrations on embryo development potential in IVF with 6 h delayed fertilization and conventional IVF.

	Treatments	Oocyte No.	Cleavage (%)	2-cell (%)	8-cell (%)	Blastocyst (%)
IVF with 6 h delayed fertilization	Control	210	126 (60.0)	123 (58.6)	87 (41.4)	66 (31.4)
	5 mM	203	127 (62.6)	124 (61.1)	100 (49.3)	91 (44.8)*
	10 mM	211	126 (59.7)	119 (56.4)	90 (42.7)	73 (34.6)
Conventional IVF	Control	217	152 (70.0)	135 (62.2)	117 (53.9)	98 (45.2)
	5 mM	239	180 (75.3)	160 (66.9)	132 (55.2)	121 (50.6)
	10 mM	282	209 (74.1)	197 (69.9)	130 (46.1)	116 (41.1)

3 replicates for IVF with 6 h delayed fertilization and 4 replicates for conventional IVF.*p < 0.05 compared with control group.

Table 2. The effects of fertilization media with different glucose concentrations on mouse birth outcomes in IVF with 6 h delayed fertilization and conventional IVF.

	Treatments	NO. of transferred embryos	NO. of birth (%)	Fetal weight (g)	Crown-rump length (mm)	Tail length (mm)	Birth defects
IVF with 6 h delayed fertilization	Control	57	15 (26.3)	2.1 ± 0.1	34.0 ± 0.8	14.2 ± 0.3	0
	5 mM	71	31(43.7)*	2.1 ± 0.2	33.8 ± 0.8	14.1 ± 0.5	0
	10 mM	53	19 (35.8)	2.1 ± 0.1	33.9 ± 0.8	14.3 ± 0.3	0
Conventional IVF	Control	96	40 (41.7)	2.1 ± 0.1	33.7 ± 0.8	14.0 ± 0.4	0
	5 mM	112	43 (38.3)	2.1 ± 0.2	33.8 ± 0.7	14.0 ± 0.5	0
	10 mM	112	39 (34.8)	2.1 ± 0.2	33.7 ± 0.8	14.2 ± 0.5	0

3 replicates for IVF with 6 h delayed fertilization and 4 replicates for conventional IVF. *p < 0.05 compared with control group.

For clinical application, an increased concentration of glucose in fertilization medium must be proved to be safe and have no adverse effects on embryos which are fertilized normally. In the present study, the effects of fertilization media with different glucose concentrations on embryo development potential in conventional IVF without delay are also presented in Table 1. Fresh COCs were co-incubated with capacitated sperm in control, G-IVF PLUS with 5 mM and 10 mM glucose without the step of COCs cultured alone after COCs collection. After fertilization, embryos were cultured to blastocysts. Rates of cleavage, 2-cell, 8-cell and blastocyst were not significantly different among the three groups. The results indicate that increasing the concentration of glucose to 10 mM in G-IVF PLUS has no effects on embryo developmental potential in a conventional IVF procedure. Glucose was once believed to contribute to a 2-cell block (Schini and Bavister 1988). There is limited research on the effect of glucose on human embryos, but many studies have reported embryo development was improved in culture media without glucose (Conaghan et al. 1993; Quinn 1995). However, the effects of glucose on the early embryos are controversial. Barak et al. (1998) believed the presence of glucose in culture media improved implantation potential in ART. There was no difference in pregnancy rate between medium with 5.5 mM glucose and that without glucose (Coates et al. 1999). A monoculture system with 4.7 mM glucose supported human embryo development to blastocyst as efficiently as commercially sequential medium in human IVF (Macklon et al. 2002). Glucose in a high concentration of 5.55 mM did not affect embryo quality (Michaeli et al. 2011). Therefore, it has been thought that glucose does not inhibit early embryo development in a certain concentration, avoiding extremes (Chronopoulou and Harper 2015). The present study also supports this opinion.

Moreover, we compared the effects of fertilization media with different glucose concentrations on mouse birth outcomes when fertilization was delayed 6 h as shown in Table 2. After embryo culture, Day 4 blastocysts obtained in the three groups mentioned above were transferred. Birth rate of G-IVF PLUS with 5 mM glucose group was significantly higher than that of the control group (p < 0.05). But there were no significant differences in terms of fetal weight, crown-rump length, tail length or birth defect rate among the three groups. The results indicate that 5 mM glucose can improve the birth rate when fertilization is delayed 6 h. However, 10 mM glucose does not have an effect on birth rate.

At Day 4 of conventional IVF, blastocysts were transferred and the effects of fertilization media with different glucose concentrations on mouse birth outcomes were assessed as shown in Table 2. There were no significant differences in terms of birth rate, fetal weight, crown-rump length, tail length or birth defect rate among control, G-IVF PLUS with 5 mM and 10 mM glucose groups. These findings are consistent with those of previous studies showing that glucose at a concentration of about 5 mM did not affect pregnancy outcomes (Coates et al. 1999; Macklon et al. 2002). Our results indicate that G-IVF PLUS with 5 mM glucose is safe for IVF offspring in the mouse model and might have a good prospect of clinical application for unexpected fertilization delay.

In the present study, IVF, with fertilization delayed for 6 h was used as an oocyte aging model to investigate if glucose could improve mouse embryo development potential and birth rate when fertilization was delayed. Compared with the control group of conventional IVF, fertilization delayed 6 h significantly decreased the rates of cleavage, 8-cell and blastocyst in G-IVF PLUS (p < 0.05). The results indicate that this model can reflect a possible compromising effect on oocyte aging and embryo development potential. Rates of birth and 2-cell stage zygote were also decreased, but not significantly (P value is 0.055 and 0.44, respectively) when the control group of IVF with fertilization delayed 6 h was compared with the control group of conventional IVF. When embryos were transferred at Day 4, the number of blastocysts did not reach the maximum value. Day 4 blastocysts grew faster and had more developmental potential. Thus, birth rate may not reflect the embryo developmental potential of each group. The rate of whole blastocysts provided in Table 1 can better reflect the developmental potential of each group. Another possible reason may be due to the limitation of sample size in the present study.

For ethical reasons, mouse embryos are generally used to control the quality of human culture media in human reproductive medical centers. What’s more, mouse embryos cultured in human ART commercial culture (including Vitrolife® culture media: G-IVF, G1 and G2 series) are used as a widely accepted model to substitute human embryos in many studies (Morbeck et al. 2014; Truong et al. 2016). There are no glucose-free culture media and the composition of commercial culture media is unknown in human ART. Thus, it is difficult for us to include a negative control without glucose. If we use mouse culture media (like KOSM) in the study, it does not provide more information for whether glucose could be used to prevent oocyte aging in human ART clinical practice. To investigate the mechanism of glucose preventing oocyte aging in future studies, mouse embryos could be cultured in mouse culture media with a glucose-free control.

It should be noted that glucose was added to G-IVF PLUS adjusted to a total glucose concentration of up to 5 mM and 10 mM. In the present study, the time of blastocyst analysis was 120 h after fertilization in Table 1 while that of transfer was 96 h after fertilization in Table 2 (Truong et al. 2016). Embryo evaluation was performed at 24, 48, 96 and 120 ± 1 h after fertilization.

A large number of embryo transfers were performed in the present study, which provides a reliable support for the conclusion. However, focusing on clinical application, we did not examine the mechanisms of glucose preventing oocyte aging in the study. As an energy substance, glucose may prevent oocyte postovulatory aging by supplying energy to oocytes. Moreover, results in mice cannot be exactly equal to human oocytes or embryos. More studies should be carried out before clinical use, especially on safety. To obtain more precise information, a randomized clinical trial (RCT) with a greater population from multicenters would be proposed, and a long-term follow-up with the offspring also should be considered.

In conclusion, 5 mM glucose could improve embryo developmental potential when fertilization was delayed 6 h and did not have adverse effect on embryo quality or birth outcomes in normal IVF. It might have a good prospect of clinical application in ART.

Materials and methods

Fertilization media preparation

Glucose of G-IVF PLUS (Vitrolife, Göteborg, Sweden) was quantified using Roche Cobas chemistry analyzers (Cobas 6000 c501 modules) and Roche Cobas reagents. Glucose (Sigma, St. Louis, USA) was added to G-IVF PLUS to make total glucose concentration up to 5 mM and 10 mM after addition.

The process of IVF

All experiments involved in IVF were performed as previously described (Nagy et al. 2003). In brief, female ICR mice at 6–8 weeks old were superovulated with 10 IU of pregnant mare’s serum gonadotropin (PMSG, PROSpec, Ness-Ziona, Israel), followed by 10 IU of human chorionic gonadotropin (hCG, Livzon, Zhuhai, China) 48 h later. The next morning, mice were killed 13 h after hCG injection and cumulus-oocyte complexes (COCs) were collected from the oviductal ampullae. Sperm were obtained from cauda epididymis of adult ICR male mice and capacitated for 1 h in G-IVF PLUS at 37°C in 5% CO₂. All animal procedures were approved by the Institutional Animal Care and Use Committee of Beijing Chao-Yang Hospital, Capital Medical University, Beijing.

Gametes were then co-incubated in fertilization medium for 6 hat 37°C in 5% CO₂. After 6 h co-incubated, zygotes were washed and cultured in G-1 PLUS (Vitrolife, Göteborg, Sweden) for 48 h and then for a further 72 h in G-2 PLUS (Vitrolife, Göteborg, Sweden) at 37°C in 5% CO₂. Embryo evaluation was performed at fixed time points of 24, 48, 96 and 120 ± 1 h after fertilization. Cleavage embryos included 2-cell embryos and some broken embryos. Each group was replicated 3 ~ 4 times.

Embryo transfer

Female mice 8–12 weeks of age were mated with vasectomized males to establish pseudopregnancy. The next morning after mating, the recipients were checked for the presence of a vaginal plug. Day 4 blastocysts (96 h post-fertilization) were transferred surgically to the uteri of Day 4 pseudo-pregnant female mice. The left embryos were cultured to Day 5. Blastocysts of 10~12 were transferred to each recipient mouse, with 5~6 embryos in each uterine horn. Sixteen and twenty-eight recipients were used in IVF with 6 h delayed fertilization and conventional IVF, respectively. After birth, neonatal mice were macroscopically checked for malformations of eyes, mouse, ears, limbs and spine and crown-rump length, tail length and birth weights were measured.

Statistical analysis

Statistical analysis was performed using SPSS 23.0 statistics software. Data were expressed as means ± SD (standard deviation) or percentages. One-way ANOVA was utilized for comparisons among various groups. The chi-square test was used to analyze the differences in the case of percentage comparison. P values < 0.05 were considered to be significant.

Author contributions

Experiments performance: HPW, DPC. Study design: DPC, WHZ. Manuscript writing: DPC. Revision and support of this study: WHZ, YL.

Disclosure statement

The authors report no declaration of interest.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [81471511]; Famous Doctors Personnel Training Plan of Chao-Yang Hospital [YLCYMY-2017-21]; Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support [XMLX201825]; New Star Personnel Training Plan of Chao-Yang Hospital [WHZ CYXX-2017-19].