Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection
Özet
Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.
Abstract
Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life roughly equivalent to 40 human years. Intracytoplasmic sperm injection (ICSI) is a commonly used assisted reproductive technology in clinical practice. However, given its relatively recent emergence about 30 years ago, the long-term effects of this technique on human development remain unclear. In this study, we established the ICSI combined with embryo transfer (ET) method using a mouse model. The results demonstrated that normal mouse sperm, after undergoing in vitro culture and subsequent ICSI, exhibited a fertilization rate of 89.57% and a two-cell rate of 87.38%. Following ET, the birth rate of offspring was approximately 42.50%. Furthermore, as the mice aged, fluctuations in glucose metabolism levels were observed, which may be associated with the application of the ICSI technique. These findings signify that the mouse ICSI-ET technique provides a valuable platform for evaluating the impact of sperm abnormalities on embryo development and their long-term effects on offspring health, particularly concerning glucose metabolism. This study provides important insights for further research on the potential effects of the ICSI technique on human development, emphasizing the necessity for in-depth investigation into the long-term implications of this technology.
Introduction
Fertility issues have emerged as a major focus of medical and sociological concern, especially in modern society where declining fertility rates and the increasing severity of infertility prevalence and severity have risen to prominence. Assisted Reproductive Technology (ART) provides a wide array of possibilities to tackle these challenges, with Intracytoplasmic Sperm Injection (ICSI) commonly utilized as a therapeutic intervention.
Since Palermo reported the first successful pregnancy achieved through Intracytoplasmic Sperm Injection (ICSI) in 1992, ICSI has become a pivotal technique in Assisted Reproductive Technologies (ART)1. However, considering that ICSI has been used in clinical settings for only 30 years, a relatively brief period compared to the human lifespan, the long-term effects of ICSI, especially on offspring development, have not been extensively investigated and elucidated. At present, mice characterized by their uniform genetic background and shorter lifespan, have emerged as a widely utilized alternative model in medical research. Moreover, the mouse model can recapitulate the entire human lifespan within a compressed time frame, where one year in mice roughly corresponds to 40 years in humans2.
Over the past decade, several small-scale studies have reported that individuals conceived through ICSI may be at an increased risk of developing metabolic syndromes, such as abnormal blood sugar levels, later in life3,4. Although the evidence is not definitive, this finding has nonetheless raised serious concerns within the scientific community regarding the long-term health implications of ICSI. This situation underscores the pressing need for more rigorous assessments of ART and its long-term health consequences. Particularly in light of the limitations and ethical considerations of human studies, developing animal models that can precisely recapitulate the development of human offspring following ICSI has become increasingly crucial. In this context, the mouse ICSI-ET (Intracytoplasmic Sperm Injection-Embryo Transfer) model, owing to its capacity to mimic the human ICSI and facilitate long-term monitoring of offspring health outcomes, has become an effective tool for assessing the potential health risks of ICSI technology to offspring5.
This study aims to investigate the impact of ICSI-ET technology on a prevalent metabolic phenotype, namely offspring glucose metabolic health, by employing random blood glucose monitoring, fasting blood glucose testing, and glucose tolerance tests to assess the glucose metabolic state of mice. Random blood glucose monitoring is utilized to capture the natural fluctuations in glucose metabolism during normal physiological activities, whereas fasting blood glucose and glucose tolerance tests are employed to assess potential prediabetic states.
Protocol
Representative Results
Discussion
This study integrated mouse intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) techniques to comprehensively recapitulate human assisted reproductive technology (ART) and examine the impact of ICSI in conjunction with ET on offspring development. The application of the ICSI technique with normal mouse sperm yielded high fertilization (86.76%) and 2-cell rates (88.48%). Following ET, the birth rate of offspring mice was approximately 42.50%, indicating the robustness of the technical platform. The most notew…
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Major Project Plan of the Special Development Fund for the Shanghai Zhangjiang National Independent Innovation Demonstration Zone (ZJ2022-ZD-006), Shanghai Municipal Science and Technology Commission Targeted Funding Project (22DX1900400), Youth Program of Shanghai Municipal Health Commission (20204Y0276). the National Natural Science Foundation of China (32070849).
Materials
| 1.25% avertin (2,2,2-tribromoethanol) | Nanjing Aibei | M2960 | for anesthetization |
| Bacteriological Petri Dishes 35 x 10 mm style w/tight lid, crystal-grade virgin polystyrene, sterile | BD | 353001 | |
| Bacteriological Petri Dishes 50 x 9 mm style w/tight lid, crystal-grade virgin polystyrene, sterile | BD | 351006 | |
| Biosafety Cabinet | ESCO | class  BSC |
Aseptic operations, making culture dishes, aliquoting reagents, etc. |
| CO2 Incubator | Thermo | 8000DH | Embryo culture |
| Dissection Microscope | Olympus | SZX16 | Use in mouse embryo transfer |
| Fluorinert Fc-770 | SIGMA | F3556 | Fluorinert FC-770 is a thermally stable fully fluorinated liquid with high dielectric strength and resistivity, used as operating fluid |
| handheld glucometer | Roche | AccuChek performa | Blood glucose measurement |
| HTF | Merck | MR-070-D | The EmbryoMax Human Tubal Fluid (HTF) (1x), liquid designed for use with Mouse IVF is available in a 50 mL format and has been optimized and validated for Embryo Culture. |
| Hydraulic Microinjector | Eppendorf | CellTram 4r Oil, 5196000030 | For sperm injection |
| Inverted Microscope | Nikon | TI2-U | Micromanipulation observation host |
| KSOM | Merck | MR-020P-D | (1x), Powder, w/o Phenol Red, 5 x 10 mL |
| M2 | Merck | MR-015-D | EmbryoMax M2 Medium (1x), Liquid, with Phenol Red |
| Micromanipulator | NARISHIGE | NTX-N4 | Micromanipulation arm |
| mineral oil | SIGMA | M8410 | Mineral oil is suitable for use as a cover layer to control evaporation and cross-contamination in various molecular biology applications. |
| Needle Cutter | Nanjing Aibei | Sutter MF-800 | Use for fabricating micromanipulation needles |
| Needle Puller | Nanjing Aibei | Sutter model p-100 | Use for making micromanipulation needles |
| Piezo Drill Trip Mouce ICSI | Eppendorf | 5195000.087 | Application of ICSI injection needle. |
| Piezoelectric Micromanipulator (Membrane Breaker) | Eppendorf | Eppendorf PiezoXpert | Use of micro-pulses to break the zona pellucida and oolemma of the oocyte |
| Pneumatic Microinjector | Eppendorf | CellTram 4r Air, 5196000013 | For fixing the oocyte |
| Ready-to-use Human Chorionic Gonadotropin (Hcg) | Nanjing Aibei | M2520 | Sterilization reagent, intraperitoneal injection. 50 IU/mL |
| Ready-to-use Pregnant Mare's Serum Gonadotropin (PMSG) | Nanjing Aibei | M2620 | Sterilization reagent, intraperitoneal injection. 50 IU/mL |
| Stereomicroscope | Olympus | SZX7 | Oocyte retrieval and observation of embryo development |
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