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.
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.
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.
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.
1. ICSI in mice
2. Uterine transplantation of mouse blastocysts
3. Random blood glucose, fasting blood glucose, and glucose tolerance test
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 was no signification fluctuation in random blood glucose levels between mice born naturally and ICSI mice during the process of growth and development (Figure 2). However, male ICSI mice showed continuously high levels of fasting blood glucose throughout the experiment (Figure 3), indicating impaired fasting blood glucose homeostasis. Interestingly, female ICSI mice showed no changes compared with the control group. In GTT, the blood glucose level showed significant change between male ICSI mice and the control group (Figure 4), and there was no such difference in female mice. These results suggested that the offspring obtained using ICSI technology might suffer from diabetes, and the outcome was related to sex. Although the female mice did not show the diabetes phenotype, the overweight phenotype of both male and female ICSI mice was consistent (Figure 5).
Using the intracytoplasmic sperm injection (ICSI) protocol described here, we achieved high rates of fertilization and embryo development in mice. In a representative experiment, 89.57% of oocytes were fertilized after ICSI, as evidenced by the formation of two pronuclei. At 24 h after ICSI, 87.38% of zygotes developed to the 2-cell embryo stage. After embryo transfer to recipient females, the birth rate of live pups was 42.50% (positive results). Using the optimized intracytoplasmic sperm injection (ICSI) protocol described here, we efficiently generated offspring from mouse oocytes and sperm. Importantly, a thorough characterization of ICSI-derived animals revealed metabolic perturbations in the absence of gross developmental defects.
Compared to naturally bred controls, male but not female ICSI mice displayed impaired glucose homeostasis, evidenced by elevated fasting blood glucose (Figure 3) and reduced glucose tolerance in intraperitoneal glucose tolerance tests (Figure 4). These alterations may stem from reprogramming deficiencies during ICSI-mediated fertilization or subtle genetic/epigenetic abnormalities incurred during sperm manipulation.
Additionally, both male and female ICSI mice showed increased body weight compared to controls (Figure 5), suggesting a propensity for obesity. While the mouse ICSI technique reliably models human ICSI outcomes, the observed metabolic phenotypes underscore the need to cautiously interpret results from ICSI animal models. Comprehensive evaluation of growth, health, and behavior is imperative to determine the suitability of ICSI mice for downstream applications.
Taken together, these data illustrate the utility of the mouse ICSI protocol to efficiently generate offspring for studies, while also highlighting the importance of thoroughly evaluating phenotypic outcomes in ICSI-derived animals. The sex-specific metabolic abnormalities observed in this example underscore the need for careful interpretation of results from ICSI mouse models.
Figure 1: Diagram of the dish preparation for intracytoplasmic sperm injection in mice. The cell culture dish lid was divided into upper and lower halves. The upper half contained droplets of 10% polyvinylpyrrolidone solution for sperm placement. The lower half contained droplets of M2 medium for conducting the ICSI procedure. All droplets were covered with mineral oil to prevent evaporation. This dish setup allowed the sequential transfer of sperm and oocytes between PVP and M2 droplets to enable the ICSI process. The separated areas prevented the mixing of media during the procedure. Abbreviations: ICSI = intracytoplasmic sperm injection; PVP = polyvinylpyrrolidone. Please click here to view a larger version of this figure.
Figure 2: Weekly random blood glucose levels in naturally bred control (n = 5) and ICSI (n = 5) mice during postnatal development. Blood glucose levels were measured weekly after overnight fasting from the tail vein of control and ICSI mice using a glucometer. Data are presented as mean ± SEM. Statistical significance was assessed using a two-way ANOVA, ns, P > 0.05. Abbreviations: SEM = Standard Error of the Mean; ICSI = intracytoplasmic sperm injection; ns = not significant; NM = natural mating. Please click here to view a larger version of this figure.
Figure 3: Weekly fasting blood glucose levels in naturally bred control (n =7 for males, n = 7 for females) and ICSI-treated mice (n = 7 for males, n = 7 for females) during postnatal development. Following overnight fasting, blood glucose levels were measured weekly from the tail vein of fasted control and ICSI-treated mice using a glucometer. It was observed that male ICSI-treated mice, but not female, exhibited significantly elevated fasting blood glucose levels compared to their sex-matched controls starting from 8 weeks of age. Data are presented as mean ± SEM. Statistical significance was assessed using a two-way ANOVA, ns, P > 0.05, ***P < 0.001,****P < 0.0001). Abbreviations: SEM = Standard Error of the Mean; ICSI = intracytoplasmic sperm injection; ns = not significant; NM = natural mating. Please click here to view a larger version of this figure.
Figure 4: Intraperitoneal glucose tolerance test in naturally bred control (n = 5) and ICSI-treated male mice (n = 5) at 16 weeks of age. Following an overnight fast, mice received an intraperitoneal glucose injection (2 g/kg of body weight). Blood glucose levels were then measured at 0, 30, 60, and 120 min post injection. Male ICSI-treated mice exhibited significantly higher blood glucose levels at 30, 60, and 120 min during IPGTT compared to naturally bred control. Data are presented as mean ± SEM. Analyses were performed using two-way ANOVA. ns, P > 0.05. *P < 0.05. Abbreviations: IPGTT = Intraperitoneal glucose tolerance test; SEM = Standard Error of the Mean; ICSI = intracytoplasmic sperm injection; ns = not significant; NM = natural mating. Please click here to view a larger version of this figure.
Figure 5: Body weight tracking of naturally bred control (n = 5 males, n = 5 females) and ICSI-treated mice (n = 5 males, n = 5 females) from 4 to 20 weeks of age. Weekly measurements revealed that both male and female ICSI-treated mice exhibited significantly increased body weight compared to their gender-matched controls, beginning at 8 weeks of age. Data are presented as mean ± SEM. Analyses were performed using two-way ANOVA. ***P < 0.001. ****P < 0.0001. Abbreviations: SEM = Standard Error of the Mean; ICSI = intracytoplasmic sperm injection; ns = not significant; NM = natural mating. Please click here to view a larger version of this figure.
Oocytes | Zygotes | Fertilization rate (%) | 2-cell | 2-cell rate (%) | 4-8 cell | 4-8 cell rate (%) | Morula | Morula rate | Blastocysts | Blastocysts rate | |
Cauda sperm ICSI1 | 90 | 84 | 93.33 | 76 | 90.48 | 72 | 85.71 | 69 | 82.14 | 64 | 76.19 |
Cauda sperm ICSI2 | 80 | 72 | 90 | 62 | 86.11 | 59 | 81.94 | 53 | 73.61 | 46 | 63.89 |
Cauda sperm ICSI3 | 60 | 50 | 83.33 | 42 | 84 | 37 | 74 | 32 | 64 | 28 | 56 |
Total | 230 | 206 | 89.57 | 180 | 87.38 | 168 | 81.55 | 154 | 74.76 | 138 | 66.99 |
Table 1: Developmental outcomes following mouse ICSI. Abbreviation: ICSI = intracytoplasmic sperm injection.
No. | No. of transferred embryos | No. of birth (%) | Male | Female | Birth rate (%) |
1 | 6 | 3 | 3 | 0 | |
2 | 6 | 1 | 1 | 0 | |
3 | 5 | 2 | 0 | 2 | |
4 | 8 | 4 | 2 | 2 | |
5 | 8 | 2 | 1 | 1 | |
6 | 7 | 5 | 3 | 2 | |
Total | 40 | 17 | 10 | 7 | 42.5 |
Table 2: Birth outcomes following uterine transplantation of ICSI mouse embryos. Abbreviation: ICSI = intracytoplasmic sperm injection.
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 noteworthy finding of this study was the age-related fluctuation in glucose metabolism levels in ICSI offspring, which may be attributed to the application of the ICSI technique6.
The observed of fluctuations in glucose metabolism levels constitute a remarkable finding in this study. This finding implies potential long-term implications of the ICSI technique on the health of offspring, specifically regarding glucose metabolism7. To elucidate the reasons behind this phenomenon, we conducted a comprehensive examination of the pivotal technical steps encompassed within the ICSI procedure. We found that controlling the microinjection temperature (19 °C), ultrasound cutting duration (within 5 s), and transfer fluid volume (<0.5 cm) were important to maintain elevated embryo viability and implantation rates. Controlling the siphoning effect of the transfer pipette emerged as a critical step for precise embryo transfer. When preparing the transfer pipette, ensuring uniformity in the diameter of the tube is key to mitigating the siphoning effect and better controlling the movement of liquid within the tube. This measure is instrumental in enhancing the success rate and accuracy of embryo transfer. Furthermore, avoiding prolonged in vitro culture and overcrowding during embryo transfer also helped improve developmental competence. Employing optimized protocols, we attained a fertilization rate of 86.76% and a birth rate of 42.50% following ET, suggesting that the ICSI procedure itself did not inherently impair viability. Therefore, the fluctuations in glucose metabolism were more plausibly associated with subtle intracellular changes induced by the ART process rather than overt embryo damage.
While ART is widely regarded as safe, mounting evidence indicates that even seemingly normal ART offspring may exhibit various health risks later in life due to the unnatural reproductive characteristics and the critical periods of intervention during gamete and embryo development5,8,9,10,11,12,13. Our findings provide novel insights into the potential metabolic effects of ART and highlight the need for further studies on the long-term impacts of ART on offspring health14.
It is essential to acknowledge that this study employed a murine model; consequently, the results may not be directly transferable to human subjects. Nevertheless, given the substantial biological parallels between mice and humans, these findings retain considerable referential significance15. Furthermore, it is important to acknowledge that patients undergoing ICSI in clinical practice often have sperm abnormalities. Conversely, this study utilized normal mice for ICSI procedures, thus lacking data on ICSI offspring from mice with sperm abnormalities. However, it is reasonable to anticipate that sperm abnormalities would have a more profound impact on the long-term effects of the ICSI technique. Hence, the outcomes of this study maintain notable referential merit.
Another limitation of our study is the methodological discrepancy between our mouse model and human clinical practices in sperm immobilization. In the clinical application of ICSI, it is common practice to immobilize human sperm by physically disrupting the tail with a micropipette. However, in our mouse model experiments, we employed ultrasonic tail clipping to prepare the sperm, a method routinely used in mouse embryonic manipulation. We acknowledge that this approach may inadvertently introduce variability and potential bias in the model, especially considering the possible damage to sperm integrity and function that ultrasound may cause. This methodological difference may limit the direct translatability of our findings. In our future work, we plan to explore the use of micropipettes for disrupting the tails of mouse sperm. This adjustment aims to minimize any unintended effects on sperm and better replicate the conditions and outcomes observed in human ICSI procedures. We are committed to continuously improving our research practices to enhance the clinical relevance of our findings.
Based on the aforementioned technological platform, in future research, we will further investigate the impact of the ICSI technique on other aspects of offspring health, such as cardiovascular health and the immune system. Furthermore, longer-term follow-up studies are also imperative to comprehensively understand the long-term effects of ICSI.
The authors have nothing to disclose.
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).
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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