Minute changes to the culture environment of mouse pre-implantation embryos affects the health of the conceptus

George Koustas, Cecilia Sjoblom

University of Sydney, Department of Obstetrics and Gynaecology, Westmead Fertility Centre, Westmead Hospital, Sydney, Australia

Minute changes to the culture environment of mouse pre-implantation embryos affects the health of the conceptus

George Koustas*, Cecilia Sjoblom

University of Sydney, Department of Obstetrics and Gynaecology, Westmead Fertility Centre, Westmead Hospital, Sydney, Australia

ARTICLE INFO

Article history:

Received

Received in revised form

Accepted

Available online

Assisted reproductive technology In vitro culture Placenta Blastocyst

Objective: To investigate the consequences of removing embryos from the incubator daily, and exposing them to ambient air at 37.0 °C for short periods of time, on embryo development, gene expression and the health of the conceptus. Methods: An in vitro cross-sectional study was undertaken using CBA F1 mouse zygotes exposed to ambient air at 37.0 °C for 3, 5, and 8 min daily during their entire in vitro development. Blastocysts were analyzed for total cell numbers, apoptosis, and H19 and Igf2 expression and transferred to pseudopregnant recipient females to examine pregnancy, fetal and placental weight, and placental H19 and Igf2 expression. Results: Short exposure of pre-implantation embryos to ambient air at 37.0 °C resulted in a statistically significant reduction in blastocyst development and total cell numbers, increased apoptotic cell index, and impaired H19 and Igf2 expression in the blastocyst. Implantation rates were significantly reduced and fetuses and placentae displayed a significant reduced weight on day 18 of pregnancy. Placentae displayed significantly reduced levels of H19 and Igf2 expression. Conclusions: Exposing mouse pre-implantation embryos to ambient air at 37.0 °C, even for brief periods for routine micromanipulations is detrimental to normal embryonic development. Our results highlight the importance of how small alterations in the culture environment can have major consequences for the health of the embryo.

1. Introduction

As the success of assisted reproductive technology (ART) has improved over the last decade, concern has grown about the safety and long-term consequences for children born via these complex procedures. Methods including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and in vitro culture have been associated with impaired pre-implantation embryo development, leading to restricted fetal growth and significant phenotypic and behavioral changes in adult offspring [1-4]. Studies have demonstrated a strong association between ART and altered gene expression affecting methylation and expression of imprinted genes in the blastocyst, placenta, and fetus [5-11].

In vitro manipulation of gametes and pre-implantation embryos as part of ART may lead to an increased risk of congenital malformations and diseases caused by abnormal genomic imprinting [12-18]. This is corroborated by reports correlating abnormal genomic imprinting to epigenetic disorders, including Beckwith-Wiedman, Angleman, and Silver-Russell syndromes [17-20]. While infertility per se may signal a genetic predisposition to epigenetic instability, it does not account for all anomalies, and it is evident that reproductive technology applied to gametes and preimplantation embryos contributes to these adverse outcomes [21].

Although the effects of a range of ART techniques on embryo growth and subsequent fetal development have been extensively investigated, the effects of routine daily manipulations have not been studied. Removing the embryo culture system from the incubator and exposing it to ambient air causes minute changes to the culture environment. However, such removal and exposure may be necessary for routine procedures, such as assessing fertilization,

grading embryos, changing culture media, and transferring embryos. The time needed for these procedures (3, 5 and 8 min) is directly related to the proficiency of the individual embryologist.

Using a mouse model, we tested the hypothesis that in vitro development of the pre-implantation embryo and growth of the resulting conceptus are affected by even minute daily changes to the culture environment. We show that exposing the embryo culture to ambient air at 37.0 °C daily for very short periods throughout in vitro pre-implantation impaired embryo development. In addition, the expression patterns of the two important imprinted growth-related genes, insulin-like growth factor (Igf2) and H19, were altered in embryos and placentas.

2. Materials and methods

2.1. Animals and embryo collection

Animal procedures followed the Australian Code of Practice for the Use of Animals for Scientific Purposes (7th edition) under ethics approval from The University of Sydney, Sydney West Area Health Service Animal Ethics Committee (Approval Number 4155).

All handling and culture media used in this study were from Vitrolife AB, Vastra Frolunda, Sweden, which are commonly in IVF. Pre-pubertal (3–4 week) C57 × CBA, F1 (Monash Animal Services, Monash University, Victoria, Australia) female mice were injected intraperitoneally with 5 IU pregnant mare’s serum gonadotropin (PMSG; Folligon, Intervet, Australia Pty. Ltd., Bendigo, Australia) followed 48 h later by intraperitoneal injection of 5 IU human chorionic gonadotropin (hCG; Chorulon, Intervet, Victoria, Australia) and placed one per cage with 8- to 10-week-old C57 × CBA, F1 males. Female mice were sacrificed by cervical dislocation at 21 h post hCG (day 1 of pregnancy). Pronucleate oocytes were recovered in G-MOPS? PLUS and denuded using 80 IU HYASE?10× in 100 μL drops under OVOIL? and washed in 50 μL drops of G-MOPS? PLUS under OVOIL?.

2.2. Experimental design and embryo culture

Mouse zygotes were randomly allocated into experimental and control groups. These groups included i) embryos not removed from the incubator other than for change of culture medium [day 3, (26.0±2.1) s; control), ii) embryos removed from the incubator for 3 min daily, iii) 5 min daily, and iv) 8 min daily. Whenever embryos were removed from the incubator, they were held on a controlled heated stage assuring (37.0±0.2) °C in the culture medium drop. Embryos were cultured in groups of 10 in 20 μL drops of G-1?PLUS culture medium under OVOIL? for 48 h. On day 3 of development, the embryos were transferred in 20 μL drops of G-2?PLUS blastocyst medium until day 4 of development at the blastocyst stage. All cultures were performed at 37.0 °C and 6% CO2.

2.3. Cell number, allocation, and apoptosis

Blastocyst total cell, trophectoderm (TE), and inner cell mass (ICM) numbers and apoptosis were determined as described previously [22]. Blastocysts were washed three times in phosphatebuffered saline containing polyvinylpyrrolidone (PBS)/PVP (1 μg/mL), fixed in 3.7% paraformaldehyde in PBS, pH 7.3, overnight at 4.0 °C, then washed again in PBS/PVP, and stored at 4.0 °C until analysis. Blastocysts were permeabilized in 0.5% Triton X-100 (Sigma-Aldrich Chemical Co., St. Louis, MO) for 1 h at room temperature, washed three times in PBS/PVP, and incubated in TUNEL reagent (Roche Molecular Biochemicals, Indianapolis, IN) for 1 h at 37.0 °C in the dark. A TUNEL-negative control was obtained by omitting Terminal deoxynucleotidyl transferase (TdT) from the labeling mix. Embryos were then washed in PBS/PVP and counterstained with the fluorochrome propidium iodide (PI; 50 mg/mL in PBS; Sigma-Aldrich Chemical Co.) for 60 min at room temperature in the dark. To avoid background staining from cytoplasmic RNA, RNase A (60 mg/mL; Sigma-Aldrich Chemical Co.) was added to the PI labeling mix. After extensive washing, the embryos were mounted under cover-slips in mounting medium (Invitrogen Molecular Probes, Eugene, OR). Slides were sealed with nail varnish and stored at 4 °C until confocal analysis. Fluorescence was detected using a Leica scanning confocal microscope (Leica TC2 SP2 Laser Scanning Spectral Confocal Microscope, Leica Microsystems, Wetzlar, Germany). Blastocysts were scanned in two channels to detect PI and TUNEL. At least 18–20 optical sections were taken through each blastocyst at 3- to 4-μm intervals. TE and ICM were distinguished according to morphology and position described previously [23].

2.4. RNA preparation, cDNA synthesis and real time qRTPCR

To achieve a homogenous population for RNA purification, total RNA isolation from pools of 30 blastocysts or placental tissue was obtained using the RNeasy Micro Kit Plus (Qiagen Pty. Ltd., Valencia, CA) following the manufacturer’s instructions. First strand cDNA libraries were generated using the SuperScript? III First-Strand Synthesis SuperMix for real-time qRT-PCR (Invitrogen Molecular Probes), according to the manufacturer’s protocol. Six microliters of total RNA were combined with 10 μL of 2× RT Enzyme mix (including oligo-dT20, random hexamers, MgCl2, and deoxynucleotide triphosphates [dNTPs]) and 2 μL of RT Enzyme mix and incubated at 50.0 °C for 30 min, followed by incubation at 25.0 °C for 10 min. Reactions were terminated by denaturing the reverse transcriptase enzyme at 85.0 °C for 5 min, following incubation on ice for 2 min. Finally, 1 μL (2 U) of Escherichia coli RNase H was added, and reactions were incubated at 37.0 °C for 20 min.

Quantitative gene expression analyses of genes H19 and Igf2 were

performed, using the housekeeping gene Sdha as an endogenous control. Each primer was first tested for amplification on cDNA standards from day 17 mouse placenta. Real-time qRT-PCR was performed on the Corbett Rotorgene 6000 (Corbett Research Pty. Ltd, Sydney, Australia) by TaqMan? technology using TaqMan? assays (Gene IDs; Sdha: Mm_01352366_m1; H19: Mm00469706_ g1; and Igf2: Mm00439565_g1). All samples were amplified in triplicate. Each 10-μL reaction contained 5.0 μL of 2 × TaqMan?Gene Expression Master Mix (ABI-Life Technologies, Carlsbad, CA), 0.5 μL of 20 × TaqMan?Gene Expression Assay (ABI-Life Technologies), 2.5 μL of nuclease-free water, and 2 μL of cDNA template. The template was replaced by nuclease-free water in the negative controls. The thermal program included 40 cycles of 95.0 °C for 10 min, followed by 40 cycles of 95.0 °C for 15 s and 40 cycles of 60.0 °C for 60 s. The relative mRNA expression was calculated by comparison, with standard curves generated from serial dilutions (1:1; 1:5; 1:25, and 1:125) of pooled day-17 mouse placental cDNA, and normalized to the average abundance of mRNA for the reference gene Sdha.

2.5. Embryo transfers

Embryo transfers were carried out as described previously [2]. Naturally ovulating 9- to 10-week-old recipient C57 × CBA, F1 female mice were mated to vasectomized 10-week-old C57 × CBA, F1 males to establish pseudopregnancy. On day 4 of development, the in vitro cultured blastocysts were transferred to pseudopregnant females on day 3 of pseudopregnancy. Five blastocysts were transferred to the left uterine horn and five control blastocysts to the right uterine horn of the mouse (i.e., test and control groups were transferred to opposite sides). On day 18 of pregnancy, implantation sites and live fetuses were determined, and fetal and placental measurements were recorded.

2.6. Statistical analysis

All statistical analyses were carried out using SPSS (SPSS, Chicago, IL). Data of blastocyst total cell numbers, allocation measurements (ICM and TE), levels of apoptosis, blastocyst, and placental gene expression, and fetal and placental weights are presented as mean±SEM. Differences in the percentages of embryos developed to two cells, blastocysts, and hatching blastocysts in different treatment groups were compared by χ2test. Total blastocyst cell numbers, levels of apoptosis in blastocyts, allocation measurements (ICM and TE), fetal and placental weights, and blastocyst gene expression levels were analyzed using oneway ANOVA test followed by post hoc Fisher’s least significance difference (LSD) test. Gene expression levels in placentae were not normally distributed and were analyzed using a nonparametric Mann–Whitney U test. P values ＜ 0.05 were considered significant.

3. Results

3.1. In vitro culture and embryo development

A total of 2 192 zygotes were cultured from the one-cell to blastocyst stage at 37.0 °C, 6% CO2in air. The number of onecell embryos that developed to two cells was not affected by daily exposure to 3 min of ambient air at 37.0 °C; however, in the 5- and 8-min groups, the first cleavage was significantly reduced compared to the control (91.8% and 91.0% vs. 95.4%; P ＜ 0.05; Table 1). Blastocyst development was affected when embryos were exposed for 8 min on a daily basis (67.5% vs. 80.0%; P ＜ 0.05; Table 1). Daily exposure of embryos to ambient air at 37.0 °C for even 3 min was associated with a significant reduction in the blastocyst hatching rate (P ＜ 0.05; Table 1).

Table 1 The in vitro development of mouse zygotes exposed to ambient air at 37.0 °C*.

3.2. Blastocyst cell number, allocation and apoptosis

Assessment of blastocyst quality by determining blastocyst cell number and allocation revealed a significant decrease in total cell numbers in all groups, compared to the control. The 8-min group displayed a significant reduction of inner cell mass (ICM; 19.91±1.03 cells vs. 15.09±1.26 cells) and trophectoderm (TE; 40.17±1.32 cells vs. 33.64±2.07 cells) compared to control blastocysts (P ＜ 0.05; Figure 1A). However, the ratio of ICM to TE in the blastocyst was not affected (P ＞ 0.05). The level of apoptosis was assessed in blastocysts. There was no significant increase in the levels of apoptosis in blastocysts in the 3- and 5-min groups (P ＞ 0.05; Figure 1B and 1 C). However, the apoptotic cell index indicated that (5.60±1.10)% of all cells in blastocysts in the 8-min group were apoptotic (P ＜ 0.05; Figure 1B and 1C).

Figure 1. Ambient air at 37.0 °C reduced total cells and induced apoptosis in blastocysts.(A) Blastocyst total cells and trophectoderm (TE) and inner cell mass (ICM) cell numbers obtained from zygotes cultured in vitro and exposed to ambient air at 37.0 °C. Data are presented as mean±SEM. (B, C) Apoptotic cells in mouse blastocysts. (B) Blastocyst from the control group not exposed to ambient air. (C) Blastocyst with yellow fragmented apoptotic cell nuclei shown by TUNEL. Data analyzed using a one-way ANOVA test, followed by post hoc Fisher’s LSD test (three replicate experiments; 20 blastocysts for each group were analyzed). *Significant difference from the control group, P＜ 0.05.

3.3. Blastocyst expression of H19 and Igf2

Exposing pre-implantation embryos for 3 and 5 min did not affect the levels of gene expression of H19 (P ＞ 0.05; Figure 2A). Embryos exposed for 8 min had significantly increased expression levels of H19, compared to the control blastocysts (P ＜ 0.05; Figure 2A). Igf2 was significantly down-regulated in all groups, compared to control blastocysts (P ＜ 0.05; Figure 2B).

Figure 2. Real time qRT-PCR quantification of H19 and IGF2 mRNA levels in blastocysts exposed to ambient air at 37.0 °C.Relative abundance of the imprinted genes (A) H19 and (B) IGF2 in blastocysts obtained from zygotes cultured in vitro and exposed to ambient air at 37.0 °C. The level of mRNA was quantified by real-time qRT-PCR and normalized to the expression of the reference gene Sdha. Data are mean±SEM and were analyzed using using a one-way ANOVA test, followed by post hoc Fisher’s LSD test (three replicate experiments; three pools of 30 blastocysts). *Significant difference from the control group, P ＜ 0.05.

3.4. Full-term development of blastocysts

The ability of blastocysts to implant and establish a pregnancy was assessed following transfer to pseudopregnant female mice. A significant reduction in implantation rate was observed in blastocysts exposed to ambient air at 37.0 °C for 8 min (52% vs 36%; P ＜0.05, Table 2). There was a significant reduction in fetal (P＜0.05; Figure 3A) and placental weight (P ＜ 0.05; Figure 3B) of conceptus resulting from implanted blastocysts in the 8-min group.

Figure 3. Fetal growth rates in utero on day 18 of pregnancy after transfer of blastocysts obtained from zygotes cultured in vitro and exposure to ambient air at 37.0 °C.Fetal outcomes following embryo transfer for (A) fetal weight and (B) placental weight. Data are presented as mean±SEM. Control, n = 21; 3 min, n = 23; 5 min, n = 23; and 8 min, n = 7 placentae and fetuses analyzed. Data were analyzed using a one-way ANOVA test, followed by post hoc Fisher’s LSD test. *Significant difference from the control group, P ＜ 0.05.

3.5. Placental expression of H19 and Igf2

Blastocysts exposed to ambient air at 37.0 °C for 3, 5, and 8 min during the pre-implantation embryo development period were transferred to pseudopregnant recipient mice to assess if the impaired H19 and Igf2 persisted post-implantation. Placental H19 expression was found to be significantly down-regulated in all groups (3, 5, and 8 min), compared to the control (P ＜ 0.05; Figure 4A). Similar to H19, Igf2 expression was significantly down-regulated in all groups (P ＜ 0.05; Figure 4B).

Figure 4. Real time qRT-PCR quantification of H19 and IGF2 mRNA levels in placental tissue.Relative abundance of the imprinted genes (A) H19 and (B) IGF2 of day-18 placentae after transfer of blastocyst obtained from zygotes cultured in vitro and exposed to ambient air at 37.0 °C. The level of mRNA was quantified by real-time qRT-PCR and normalized to the expression of the reference gene Sdha. Data are mean±SEM, and data were analyzed using Mann–Whitney U test. Control, n = 21; 3 min, n = 23; 5 min, n = 23; and 8 min, n = 7 placentae used for gene expression analysis. *Significant difference from the control group, P ＜ 0.05.

Table 2 The effect of ambient air at 37.0 °C on implantation, fetal growth and placental development on day 18 of pregnancy following day 4 blastocyst transfers to day 3 pseudopregnant female mice.

4. Discussion

The risks of ART need to be considered in light of emerging evidence of increased incidence of congenital malformations and diseases associated with abnormal genomic imprinting in children conceived through IVF and ICSI. These adverse outcomes are at least partially a consequence of compromised pre-implantation embryo development that may result from suboptimal manipulation and culture. While several studies have linked genetic alterations in pre-implantation embryo and conceptus with laboratory-induced stressors, such as the culture environment and highly invasive techniques such as ICSI, the result of minute manipulations and embryo handling have not been investigated [24].

In our study, we used short exposures (0–8 min) to ambient air to introduce minute changes to the embryo culture system by removing embryos in culture from a controlled incubator environment for short periods daily. The effects of these minute changes were then assessed through analyses of embryo developmental capacity and expression profiles of two growth-related imprinted genes in embryos and placental tissue.

We found that the first cleavage of zygotes and their potential to form blastocysts were decreased when embryos were removed from the incubator for short times daily (5 and 8 min). Blastocysts in the culture systems that were exposed to daily 8-min periods of ambient air had a lower number of cells and an increased incidence of apoptosis. The viability of blastocysts, as determined by their ability to expand and fully hatch, was decreased with exposure to ambient air at 37.0 °C, for as short a time as 3 min daily. The ability of blastocysts to hatch is of great significance, because it affects subsequent implantation and establishment of pregnancy [25]. A blastocyst’s failure to hatch, decreased cell numbers, and increased apoptosis are associated with suboptimal embryo culture conditions [25-29].

Removing a carbonate-buffered embryo culture system from a CO2-containing environment inevitably could change the culture pH. The pH regulatory mechanisms in the embryo are activated immediately after fertilization and pronuclear formation, and the cleavage-stage embryo efficiently regulates the diverse alkaline and acidic loads it encounters during its passage through the fallopian tube to the uterus [30-32]. However, the pH of in vivo transition changes gradually and does not exhibit the specific fluctuating pattern found in an in vitro culture system, which is a rapid alkaline change induced by a short exposure to ambient air, followed by an equally rapid acidic change after being returned to the incubator. Similarly, small pH fluctuations in the culture environments brought on by increased incubator door openings and daily observations of human pre-implantation embryos have been associated with reduced quality and developmental rates of blastocysts [33]. Our data could suggest that even the smallest perturbations in the culture system may adversely contribute to developmental competence and viability of embryos.

Gene expression analysis of expanded blastocysts resulting from daily exposure of the culture system to ambient air for even short times (3, 5, and 8 min) resulted in significant decreases in Igf2 expression, while exposure for 8 min was associated with a significant increase in H19 expression. The imprinted genes H19 and Igf2 play a critical role in regulating fetal and placental development and in the pathogenesis of imprinting disorders observed in human, which a main reason why they were selected for analysis in this study. In vitro culture and IVF are associated with altered expression of Igf2 and H19 in blastocysts, indicating that these genes are extremely sensitive to in vitro manipulation, compared with other imprinted genes [7,34]. Suboptimal culture conditions have also been linked with perturbed and -even complete loss of H19 expression in blastocysts [35-37].

Embryos were transferred to pseudopregnant recipient mice, and our results showed that the conceptus resulting from the exposed embryos were significantly affected. Eight-minute exposure was associated with significantly decreased implantation rate as well as fetal and placental weight. This finding is of particular importance, because correct development and function of the placenta are crucial for subsequent fetal development and fetal programming [38]. In addition, the placenta, which facilitates physiologic intrauterine development and fetal growth, may be a good indicator of fetal health [39]. It is therefore not surprising that minute changes in the environment during in vitro culture, which likely undermined the

adequate development of the placenta, led to fetuses with body weights that averaged 22% less than the control.

Further gene expression analysis of conceptus from embryos with daily exposure to ambient air (3, 5, and 8 min) showed aberrant expression of H19 and Igf2 in the placentae. Other studies have found that IVF and in vitro culture of mouse embryos affect not only the expression of H19 and Igf2 in placentae but can lead to impaired expression of a large number of placental genes. H19 expression is limited to the TE cells of the blastocyst, so these cells might be more sensitive to epigenetic disturbances caused by in vitro culture and manipulation [6,40]. Pre-implantation effects on H19 persist post-implantation in a tissue-specific manner, because of a loss of specific methylation of the H19 differentially methylated domain in blastocysts and placentae. Since H19 and Igf2 are regulated by the same ICR in opposite ways methylation analysis of ICR would clarify contradictory upregulation of H19 at the blastocyst stage and down-regulation in placentae [41].

This effect indicates a possible inability of TE cells to restore genomic imprints in the resulting placenta, whereas it has been suggested that ICM cells exhibit robust mechanisms that protect imprinting in the embryo. The sensitivity of TE cells could result from their closer contact with culture medium and the fact that they are the first cells to differentiate in the developing embryo. In addition, studies associate the inability of TE cells to restore methylation errors in the placenta to a lack of DNA methylatransferases, which maintain methylation in imprinted genes [42-44]. The function of H19 in placental development is currently unknown, although H19 is associated with the oppositely imprinted gene Igf2, which is a major regulator of fetal growth [39]. Abnormal expression of H19 and Igf2 is closely linked with aberrant fetal and placental growth, and abnormal regulation of the H19 promoter or deletion of H19 results in abnormal expression of Igf2, creating disturbed regulation of fetal and placental growth [45-47]. Loss or deregulation of Igf2 has been linked with up-regulation or loss of expression of H19, resulting in offspring with affected weight in both human and mouse [39,48-55].

Together the results of our study show that there is a domino effect, in which even minute changes to the mouse embryo cultures significantly affect the developmental competence and viability of the embryos, leading to blastocysts with lower cell numbers and altered gene expression. While these changes do not prevent embryos from implanting and forming a conceptus, the fetal and placental weights were significantly decreased and the genetic perturbations were memorized in the conceptus. Numerous studies in animal models have demonstrated that many components of ART have a negative impact on the offspring, including impaired growth and perturbed gene expression in the conceptus and offspring as a result of IVF and in vitro culture; oxygen tension during culture; exposure to fluorescent light and sunlight; disturbances in embryo metabolism; and ICSI [2,24,56-61]. More concerning, several reports in humans demonstrate that placentae from growth-restricted pregnancies exhibit reduced expression of Igf2 [62-64]. Also, both singleton and twin babies conceived through ART exhibit preterm birth and low birth weight [65-68].

Taken together, these reports and the results of this study suggest that IVF laboratory methods may be correlated with aberrant H19 and Igf2 expression and reduced birth weight of children conceived through ART and that individual embryologists’ proficiency may play a role as well.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

The authors thank Dr Laurence Cantrill for helping with the laser scanning confocal microscopy at Kids Research Institute, University of Sydney. The study was supported by funds from Westmead Institute of Reproductive Medicine (WIRM), Westmead Hospital, Hawkesbury Rd, Westmead, NSW.

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ment heading

10.1016/j.apjr.2016.06.015

*Corresponding author: George Koustas, University of Sydney, Department of Obstetrics and Gynaecology, Westmead Fertility Centre, Westmead Hospital, Sydney, Australia.

E-mail: gkoustas@westmeadivf.com

Foundation project: The study was supported by funds from Westmead Institute of Reproductive Medicine (WIRM), Westmead Hospital, Hawkesbury Rd, Westmead, NSW.

H19/Igf2