Özet

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Published: May 17, 2024
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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

The protocol of ICSI-ET described below follows the guidelines and has been approved by the Animal Ethical Review of the Shanghai Institute of Planned Parenthood research. Safety Procedures: Always wear appropriate personal protective equipment (PPE) when handling chemicals or biological materials. Use of Hoods: Perform all procedures involving volatile chemicals or aerosol generation within a certified fume hood or biosafety cabinet. Use female mice (6-8 weeks old B6D2F1 strain) for the superovulation procedure. <p …

Representative Results

In our laboratory, we have achieved a fertilization rate of 89.57% and a 2-cell rate of 87.38% using ICSI with epididymal caudal sperm in mice. The birth rate of offspring following ET is approximately 42.50%. Remarkably, all the fertilization rates, 2-cell rates, and offspring birth rates are comparable to the levels achieved in human ART, enabling a comprehensive simulation of different stages of human ART techniques in mice. Further details are provided in Table 1 and Table 2. There w…

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 Equation 1 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

Referanslar

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Bu Makaleden Alıntı Yapın
Tang, J., Zhang, B., Tang, C., Wang, X., Hua, M., Zhou, Y., Shi, H., Zhang, T. Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection. J. Vis. Exp. (207), e66212, doi:10.3791/66212 (2024).

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