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).

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
<ol>
	<li>Superovulation of mice
	<ol>
		<li>Administer an intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) at a dose of 7.5 IU per mouse at around 20:00 on the first day.</li>
		<li>Administer an intraperitoneal injection of human chorionic gonadotropin (HCG) at a dose of 7.5 IU per mouse at around 20:00 (48 hours after PMSG injection) on the third day.</li>
	</ol>
	</li>
	<li>Prepare HTF medium into 1 mL aliquots and leave them uncovered for overnight equilibration at 37 &#176;C in a 5% CO2 incubator on the same day of HCG injection in female mice.</li>
	<li>Preparation of ICSI fertilization droplets
	<ol>
		<li>On the same day of HCG injection in female mice, divide a 50 x 9 mm cell culture dish lid into upper and lower halves. Use the upper half to create 4-6 droplets of 5 &#181;L each, using 10% polyvinylpyrrolidone (PVP) for sperm placement. Use the lower half to create 8-10 droplets of 5 &#181;L each, using M2 medium for the ICSI procedure.</li>
		<li>Cover the PVP and M2 medium droplets with approximately 3 mL of mineral oil (Figure 1).</li>
	</ol>
	</li>
	<li>Preparation of culture dishes
	<ol>
		<li>On the same day of HCG injection in female mice, dispense 12 drops of KSOM medium (MR-020P-D) of 20 &#181;L evenly in a 35 x 10 mm cell culture dish. Cover the drops with approximately 3 mL of mineral oil.</li>
	</ol>
	</li>
	<li>Sperm harvesting:
	<ol>
		<li>Euthanize male mice by cervical dislocation at approximately 9:00 am on the fourth day (13 h after HCG injection).</li>
		<li>Isolate the epididymal cauda and thoroughly clean it to eliminate any adipose tissue and blood remnants by gently blotting with sterile gauze and rinsing in prewarmed HTF medium.</li>
		<li>Cut open the cauda epididymis to allow the sperm to freely swim out into the HTF medium. Gently squeeze the cauda using forceps to release the sperm, collect them in preequilibrated HTF tubes, and keep them uncovered in a 37 &#176;C, 5% CO2 incubator for sperm capacitation for further use.</li>
	</ol>
	</li>
	<li>Oocyte retrieval
	<ol>
		<li>Euthanize female mice stimulated for oocyte retrieval by cervical dislocation at around 9:30 am on the fourth day. Carefully make an incision along the midline of the abdominal skin and muscle layer to expose the abdominal cavity.</li>
		<li>Use fine forceps to gently isolate tissue from both sides of the oviducts, especially the swollen ampulla regions, and transfer it into M2 medium in a sterilized culture dish. Repeat this procedure until the oviducts from all the designated mice are collected.</li>
		<li>Under a dissecting microscope, use a 1 mL syringe needle to puncture the transparent section of the swollen ampulla of the oviduct gently, facilitating the release of cumulus-oocyte complexes (COCs) into the medium.</li>
		<li>Using the same syringe, carefully pick and transfer the COCs into a new dish containing 100 &#181;L of fresh M2 medium. Repeat this step until all COCs are successfully transferred from the original oviduct segments.</li>
		<li>Wipe away blood and tissue fluids, and under a dissecting microscope, tear open the ampulla of the oviduct using a 25 G needle, resulting in the release of numerous COCs.</li>
		<li>Remove the oviduct and tissue fragments, transfer the COCs into droplets of M2 medium containing hyaluronidase (HY), and prewarm them to 37 &#176;C.</li>
		<li>After incubation for approximately 5 min, carry out gentle pipetting to facilitate the separation of cumulus cells and oocytes. Transfer the metaphase II (MII) oocytes from the HY-containing solution to fresh M2 medium; then, perform the sequential transfer into multiple droplets of M2 medium.</li>
		<li>After 2-3 rounds of washes, transfer the oocytes to droplets of KSOM culture medium and keep them in a 37 &#176;C, 5% CO2 incubator for further use.</li>
	</ol>
	</li>
	<li>Around 10:00 am on the fourth day, sonicate 100 &#181;L of the capacitated sperm (see step 1.5.2.) for 5 s to separate the heads from the tails at a frequency of 46 kHz, with a power setting of 50 watts. 
	NOTE: When sonicating sperm to truncate the tail, be cautious to control the time, which should not exceed 5 s. A few seconds after sonication, sperm can be observed under an inverted microscope; stop sonicating when approximately half of the sperm show truncated tails.</li>
	<li>ICSI procedure
	<ol>
		<li>Use a commercially available micromanipulation needle with a flat tip and an inner diameter of 6 &#181;m, set at a 25&#176; angle, for the injection. Fill the needle with the operating fluid with the filled portion measuring a length of 0.5 cm inside the needle. Ensure that the level of the fluid reaches ~0.5 cm. 
		NOTE: Maintain room temperature at 19 &#176;C during injection to avoid high temperatures that may reduce the survival rate of fertilized oocytes. The precision in ensuring the fluid level reaches 0.5 cm is crucial for maintaining the integrity of the injection process and ensuring the viability of the oocytes.</li>
		<li>Install the injection needle on the right arm of the micromanipulator, securely fixed by tightening the holding cap. Install a commercially available flat-tipped micromanipulation holding pipette designed for mouse oocytes on the left arm of the micromanipulator, and position the injection needle and the holding pipette in the microscope&#39;s field of view.</li>
		<li>Add 1 &#181;L of sonicated sperm to a droplet of PVP solution using a pipette.</li>
		<li>Place 20 MII oocytes in a droplet of M2 medium.</li>
		<li>Use the injection needle to aspirate 10-20 sperm heads in PVP solution. Then, move the injection needle into the droplet containing the oocytes.</li>
		<li>Directly determine the position of the metaphase spindle within the oocyte under a polarization microscope to ensure that the spindle apparatus is not on the side of injection, with the polar body typically located at either the 12 o&#39;clock or 6 o&#39;clock position relative to the oocyte&#39;s orientation.</li>
		<li>Keep the oocyte in contact with the bottom of the culture dish. Adjust the focal plane so that the zona pellucida of the oocyte and the injection needle are in the same plane.</li>
		<li>Upon contact of the injection needle with the zona pellucida, apply a slight negative pressure to the injection needle, drawing in a portion of the zona pellucida.</li>
		<li>Simultaneously activate piezo pulses with an intensity setting of 5 and a frequency of 1, effectively creating a perforation in the zona pellucida and allowing the injection needle to pass through.</li>
		<li>Advance the injection needle into the perivitelline space, pushing the sperm head to the tip of the needle. Insert the injection needle into the oocyte until it reaches the opposite side of the zona pellucida.</li>
		<li>Use piezo pulses set to an intensity of 1 and a frequency of 1 to create a small pore in the plasma membrane, ensuring the sperm head is injected into the ooplasm with minimal entry of PVP and optimal preservation of the oocyte&#39;s integrity.</li>
		<li>Repeat step 1.8.11 until all 20 oocytes are injected, followed by a 15 min incubation at 19 &#176;C.</li>
		<li>Transfer the surviving oocytes to KSOM medium and place them in the incubator. Observe 2-cell, 4-cell, morula, and blastocyst formation rates. 
		NOTE: Timing for intracytoplasmic single sperm injection (ICSI) into the oocyte should be controlled, aiming to complete the procedure within 14-18 h after the injection of HCG.</li>
	</ol>
	</li>
</ol>
2. Uterine transplantation of mouse blastocysts
<ol>
	<li>Preparation of vasectomized male mice 
	NOTE: Use ICR male mice aged 6-8 weeks for the study.
	<ol>
		<li>Induce anesthesia by intraperitoneal injection of 1.25% 2,2,2-tribromoethanol at a dosage of 0.2 mL/10 g of body weight. The absence of any pain response indicates successful anesthetization. 
		NOTE: While the current protocol does not include the application of vet ointment to the eyes of anesthetized animals, it is recommended as a best practice to prevent eye dryness and ensure the health and comfort of the animals during procedures. Throughout the surgical and recovery process, all surgical instruments were sterilized before use, and the surgical area was prepared using antiseptic solutions to ensure a clean operating environment.</li>
		<li>To safeguard against potential stress or injury, house the mice that have undergone surgery individually during the initial recovery phase.</li>
		<li>Administer buprenorphine at a dosage of 0.05 mg/kg of body weight to ensure optimal pain relief. 
		NOTE: To ensure the well-being of the mice, closely monitor the mice during the recovery period and do not leave them unattended until they regain sufficient consciousness to maintain sternal recumbency.</li>
		<li>Prepare the surgical site at the lower abdominal region, superior to the testes. Remove the fur and disinfect with povidone-iodine. Using sterile scissors, make a 1 cm longitudinal incision along the linea alba. Gently remove any blood with sterile, dust-free paper and proceed to bluntly dissect the abdominal muscles along the linea alba and access the abdominal cavity.</li>
		<li>Using curved forceps, gently grasp and expose the left testis and its associated fat pad. Identify the vas deferens and carefully isolate it using pointed forceps, creating a 10 mm segment for manipulation.</li>
		<li>Sterilize the curved forceps by holding them in the outer flame of an alcohol lamp for several seconds. Gently grasp the vas deferens, causing it to become flattened and whitened due to cauterization. Create a second cauterization point 3-5 mm distal to the first. Using sterile surgical scissors, excise the segment of the vas deferens between the two cauterization points.</li>
		<li>Carefully inspect the cauterized ends of the vas deferens, ensuring that a short, flattened, whitened segment remains. Using curved forceps, gently return the testis and its associated fat pad to the abdominal cavity.</li>
		<li>Repeat the procedure described in step 2.1.6 on the contralateral (right) side.</li>
		<li>Close the surgical site using sutures, typically placing one stitch in the muscle layer and three stitches in the skin. Ensure proper alignment of the surgical incision and disinfect with povidone-iodine. Transfer the animal to a heating pad for observation, monitoring for 10-15 min until the animal regains consciousness. Once recovered, return the animal to its housing cage. 
		NOTE: Following vasectomy surgery, mice require a 14-day recovery period, followed by a 1-week trial mating experiment.</li>
		<li>During this week, continuously mate the vasectomized males with females and observe the pregnancy status of all females presenting with copulatory plugs. Successful mating is indicated by the presence of a yellowish-white, blocky vaginal plug at the vaginal orifice of the female mouse.
		<ol>
			<li>Pair the mice between 4:00 PM and 6:00 PM on the first day and check the vaginal plugs between 7:00 AM and 9:00 AM on the second day. Consider females with vaginal plugs to be in a pseudopregnant state at 0.5 days and use them for subsequent embryo transfer experiments. Return females without vaginal plugs to the recipient group. 
			NOTE: These females without vaginal plugs can continue to be used for mating to prepare pseudopregnant females. Ensure that all plugged females do not become pregnant; only then can the vasectomized males be used for formal experiments.</li>
			<li>While vasectomized males can mate daily, allow for 1 day of rest between mating sessions. 
			NOTE: Based on our experience, vasectomized males can maintain a high copulatory plug rate for over a year. However, vasectomized males that fail to produce copulatory plugs in mating females for 4-6 consecutive attempts should be removed from the study.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of pseudopregnant females
	<ol>
		<li>Generate pseudopregnant female mice by mating ICR female mice (aged 7-10 weeks, weighing over 25 g) with vasectomized male mice (3-6 months old) at a 1:1 ratio. 
		NOTE: The vasectomized males were used to stimulate the uterine cervix of the female mice, resulting in the activation of the corpora lutea.</li>
	</ol>
	</li>
	<li>Preparation of the pipette for transferring blastocysts
	<ol>
		<li>Take the embryos to be transplanted out from the incubator and transfer them into a droplet of M2 medium at a temperature of 37 &#176;C.</li>
		<li>Use a transfer pipette to aspirate a small volume of liquid, draw a small amount of air, and aspirate 6-10 embryos. Finally, aspirate another small volume of air to seal the pipette. 
		NOTE: The length of the fluid in the transfer pipette should not exceed 0.5 cm.</li>
	</ol>
	</li>
	<li>Uterine transplantation (blastocysts)
	<ol>
		<li>Weigh the pseudopregnant female mice (2.5 days post mating) and anesthetize them by intraperitoneal injection of 1.25% 2,2,2-tribromoethanol at a dose of 0.2 mL/10 g of body weight. Gently press the ear with forceps.</li>
		<li>After administering anesthesia to the mice and ensuring asepsis through alcohol disinfection, make a midline longitudinal incision on the ventral abdomen. Taking advantage of the loose subcutaneous tissue, shift the incision to the left or right side as required for the transplant. Use small scissors to perform blunt dissection of the abdominal wall muscle layer at ~0.5 cm lateral to the lumbar spine to access the peritoneal cavity.</li>
		<li>Using atraumatic forceps, carefully exteriorize the ovaries, oviducts, and uterus.</li>
		<li>Using the tip of a syringe needle, make a small, upward-angled incision on the uterine wall below the utero-tubal junction, avoiding blood vessels. This incision provides access to the uterine cavity; gently insert the oviduct 2-3 mm through the small hole.</li>
		<li>Carefully expel the small air bubble at the end of the transfer pipette while cautiously watching the transfer pipette during the slow injection of the embryos. Once the central portion of the culture medium is confirmed to be expelled, halt the procedure and gently remove the transfer pipette.</li>
		<li>Reposition the ovaries, oviducts, and uterus back into the abdominal cavity. Suture the muscle layer and skin separately. Place the mice on a heating pad until they recover from anesthesia. To ensure the well-being of the mice, closely monitor the mice during the recovery period and do not leave them unattended until they regain sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
	<li>Observation of pregnancy
	<ol>
		<li>Following the surgery, observe the mice for 18-21 days to monitor the number of offspring born. In case of dystocia (difficult birth), perform a cesarean section. Allow the newborn mice to be fostered by lactating dams of the same age group.</li>
	</ol>
	</li>
</ol>
3. Random blood glucose, fasting blood glucose, and glucose tolerance test
<ol>
	<li>Tracking random blood glucose levels during mouse growth
	<ol>
		<li>Since the birth of the mice, monitor the random blood glucose level every 4 weeks at 9 am after a good night of feeding and fun.
		<ol>
			<li>Puncture the mouse tail tip to obtain tail vein blood.</li>
			<li>The first drop of blood has a higher sugar measurement value due to the presence of the tissue fluid mixed with it. Therefore, wipe the first drop of blood using clean absorbent paper gently.</li>
			<li>Measure the blood glucose from the second drop (~4 &#181;L) from the tail vein with a handheld glucometer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tracking changes in fasting blood glucose and glucose tolerance during mice development 
	NOTE: Considering that the first blood sampling time point is 15 min, all mice need to be injected within 15 min. Since each mouse injection took 20-30 s, to ensure that the injections are completed within this timeframe, the maximum number of mice that can be used in each test cannot exceed 30.
	<ol>
		<li>Perform the fasting blood glucose and glucose tolerance tests every 4 weeks after birth in overnight-fasted mice.
		<ol>
			<li>On the first day, prepare fresh irradiated shavings bedding, clean cages, lids, and water bottles. Transfer the mice into a fresh cage without food. Add do not feed tags to the cages.</li>
			<li>Prepare sufficient 10% glucose injection (sterile and pyrogen-free) and 1 mL syringes.</li>
			<li>Ensure that the mice in each group fast for 16 h, from 5:00 pm on day 1 to 9:00 am on day 2, but allow free access to water. Measure the fasting blood glucose levels as described in steps 3.1.1.1-3.1.1.3 using a glucose meter.</li>
		</ol>
		</li>
		<li>Weigh the mice and mark their tails to facilitate rapid identification. Prepare glucose syringes with 10 &#181;L/g of mouse weight.</li>
		<li>At 30 s intervals, inject 10 &#181;L/g glucose into the intraperitoneal cavity of the mouse. 
		NOTE: Special attention needs to be paid to injection techniques here to avoid inaccurate liquid injection volume. The key points of operation are as follows:
		<ol>
			<li>Immobilize the mouse and restrain the tail with one hand. Keep the head slightly lower than the buttocks so that the organs in the abdominal cavity will shift towards the head, leaving enough injection space.</li>
			<li>Position the needle between the midline and the hip bone, insert it through the skin at a 45-degree angle, then gently guide the needle deeper into the abdominal cavity while keeping it aligned parallel to the skin surface for ~0.5 cm. 
			NOTE: Pay attention to any resistance during injection to determine whether the internal organs are accidentally punctured.</li>
			<li>Eject the syringe into the abdominal cavity.</li>
			<li>Record the time when the injection started and use a timer to control the injection rate. Set the timer to 90 s for three mice to avoid losing time in handling the timer.</li>
		</ol>
		</li>
		<li>Take the blood glucose measurement from the tail vein at the desired intervals (0 prior to glucose administration, 15, 30, 60, 90, and 120 min after the glucose loading). 
		NOTE: Subsequent blood glucose testing should also take 20-30 s for each mouse.</li>
		<li>Return the mice to their home cages equipped with food and water after the experiment.</li>
	</ol>
	</li>
</ol>

ICSI-ET Mouse Model to Study Long-Term Impacts on Glucose Metabolism

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The authors have no conflicts of interest to disclose.

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...

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.

<table><tbody><tr><td>1.25% avertin (2,2,2-tribromoethanol)</td><td>Nanjing Aibei</td><td>M2960</td><td>for anesthetization</td></tr><tr><td>Bacteriological Petri Dishes 35 x 10 mm style w/tight lid, crystal-grade virgin polystyrene, sterile</td><td>BD</td><td>353001</td><td></td></tr><tr><td>Bacteriological Petri Dishes 50 x 9 mm style w/tight lid, crystal-grade virgin polystyrene, sterile</td><td>BD</td><td>351006</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>ESCO</td><td>class &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66212/66212eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; BSC</td><td>Aseptic operations, making culture dishes, aliquoting reagents, etc.</td></tr><tr><td>CO2 Incubator</td><td>Thermo</td><td>8000DH</td><td>Embryo culture</td></tr><tr><td>Dissection Microscope</td><td>Olympus</td><td>SZX16</td><td>Use in mouse embryo transfer</td></tr><tr><td>Fluorinert Fc-770</td><td>SIGMA</td><td>F3556</td><td>Fluorinert FC-770 is a thermally stable fully fluorinated liquid with high dielectric strength and resistivity, used as operating fluid</td></tr><tr><td>handheld glucometer</td><td>Roche</td><td>AccuChek performa</td><td>Blood glucose measurement</td></tr><tr><td>HTF</td><td>Merck</td><td>MR-070-D</td><td>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.</td></tr><tr><td>Hydraulic Microinjector</td><td>Eppendorf</td><td>CellTram 4r Oil, 5196000030</td><td>For sperm injection</td></tr><tr><td>Inverted Microscope</td><td>Nikon</td><td>TI2-U</td><td>Micromanipulation observation host</td></tr><tr><td>KSOM</td><td>Merck</td><td>MR-020P-D</td><td>(1x), Powder, w/o Phenol Red, 5 x 10 mL</td></tr><tr><td>M2</td><td>Merck</td><td>MR-015-D</td><td>EmbryoMax M2 Medium (1x), Liquid, with Phenol Red</td></tr><tr><td>Micromanipulator</td><td>NARISHIGE</td><td>NTX-N4</td><td>Micromanipulation arm</td></tr><tr><td>mineral oil&amp;nbsp;</td><td>SIGMA</td><td>M8410</td><td>Mineral oil is suitable for use as a cover layer to control evaporation and cross-contamination in various molecular biology applications.</td></tr><tr><td>Needle Cutter</td><td>Nanjing Aibei</td><td>Sutter MF-800</td><td>Use for fabricating micromanipulation needles</td></tr><tr><td>Needle Puller</td><td>Nanjing Aibei</td><td>Sutter model p-100</td><td>Use for making micromanipulation needles</td></tr><tr><td>Piezo Drill Trip Mouce ICSI</td><td>Eppendorf</td><td>5195000.087</td><td>Application of ICSI injection needle.</td></tr><tr><td>Piezoelectric Micromanipulator (Membrane Breaker)</td><td>Eppendorf</td><td>Eppendorf PiezoXpert</td><td>Use of micro-pulses to break the zona pellucida and oolemma of the oocyte</td></tr><tr><td>Pneumatic Microinjector</td><td>Eppendorf</td><td>CellTram 4r Air, 5196000013</td><td>For fixing the oocyte</td></tr><tr><td>Ready-to-use Human Chorionic Gonadotropin (Hcg)</td><td>Nanjing Aibei</td><td>M2520</td><td>Sterilization reagent, intraperitoneal injection. 50 IU/mL</td></tr><tr><td>Ready-to-use Pregnant Mare&#039;s Serum Gonadotropin (PMSG)</td><td>Nanjing Aibei</td><td>M2620</td><td>Sterilization reagent, intraperitoneal injection. 50 IU/mL</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX7</td><td>Oocyte retrieval and observation of embryo development</td></tr></tbody></table>

monitoring blood glucose in mouse offspring after intracytoplasmic sperm injection

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.

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...

Read this Scientific Article Protocol about Monitoring Blood Glucose in Mouse Offspring after Intracytoplasmic sperm injection - Embryo Transfer at JoVE.com

Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

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.

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

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 ...

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Research

JoVE Journal

Developmental Biology

447 Views.  Shanghai-MOST Key Laboratory of Health and Disease Genomics. 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.

Video: Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

Murine Round Spermatid Injection Procedure to Study Embryonic Development

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Neuroscience

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Medicine

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

Stress-induced elevations in glucose levels can confound interpretation of data derived from a conscious intraperitoneal insulin tolerance test in mice. In this article, we describe a method to acclimate the mice to handling, injections and blood sampling prior to performing the insulin tolerance test in order to limit stress-induced hyperglycemia.

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that ...

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in offspring. Animal models of MIA are used to test causality, investigate mechanisms, and develop diagnostics and treatments for these disorders. Despite their widespread use, many MIA models suffer from a lack of reproducibility and almost all ignore two important aspects of this risk factor: (i) many offspring are resilient to MIA, and (ii) susceptible offspring can exhibit distinct combinations of phenotypes. To increase reproducibility and model both susceptibility and resilience to MIA, the baseline immunoreactivity (BIR) of female mice before pregnancy is used to predict which pregnancies will result in either resilient offspring or offspring with defined behavioral and molecular abnormalities after exposure to MIA. Here, a detailed method of inducing MIA via intraperitoneal (i.p.) injection of the double stranded RNA (dsRNA) viral mimic poly(I:C) at 12.5 days of gestation is provided. This method induces an acute inflammatory response in the dam, which results in perturbations in brain development in mice that map onto similarly impacted domains in human psychiatric and neurodevelopmental disorders (NDDs).

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

Maternal infection is a risk factor for neurodevelopmental disorders. Mouse models of maternal immune activation (MIA) may elucidate infection's impact on brain development and function. Here, general guidelines and a procedure are provided to produce reliably resilient and susceptible offspring exposed to MIA.

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in ...

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for detecting the successful establishment of blood chimerism in parabionts by a caudal vein injection of glucose. Parabiotic mice were constructed. Glucose was injected into the donor mouse through the tail vein, and the fluctuation of blood glucose level was measured in both mice using a blood glucometer at different time points. Our results showed that after glucose injection, the blood glucose level in donor mice increased sharply after 1 min and decreased slowly thereafter. Meanwhile, the blood glucose level of the recipient mice peaked 15 min after injection. Similar results were obtained with Evans blue, used as a positive control for glucose. The synchronous fluctuation of blood glucose levels indicates that blood flow between the two mice was established successfully.

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...

Biology

Effect of Maternal Folic Acid and Choline Deficiencies on Offspring Health

Modifying Levels of Maternal Dietary Folic Acid or Choline to Study the Impact of Deficiencies on Offspring Health Outcomes

Maternal nutrition during pregnancy and lactation plays an important role in the neurodevelopment of offspring. One-carbon (1C) metabolism, which centers around folic acid and choline, as well as other B vitamins, plays a key role during the closure of the neural tube of the developing fetus. However, the impact of these maternal nutritional deficiencies during pregnancy on offspring health outcomes after birth remains relatively undefined. Furthermore, maternal dietary deficiencies in folic acid or choline may impact other health outcomes in offspring - making this a valuable model. This protocol aims to outline the procedure for inducing a deficiency in 1C metabolism in female mice through dietary modifications. Females are placed on diets at weaning, up to 2 months of age, for 4-6 weeks prior to mating and remain on diet throughout pregnancy and lactation. Offspring from these females can be evaluated for health outcomes. Females can be used multiple times to generate offspring, and tissues from females can be collected to measure for 1C metabolite measurements. This protocol provides an overview of how to induce maternal dietary deficiencies in folic acid or choline to study offspring health outcomes.

Author Spotlight: Studying the Impact of Maternal Dietary Deficiencies on Long-Term Offspring Health Outcomes

This protocol describes the method for reducing dietary intake of folic acid or choline in female mice prior to pregnancy with the objective of investigating the impact of maternal diet on offspring health outcomes.

Maternal nutrition during pregnancy and lactation plays an important role in the neurodevelopment of offspring. One-carbon (1C) metabolism, which centers ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits is causal remains elusive. Moreover, whether these deficits are mediated by an increase in glucose levels in cerebrospinal fluid (CSF) and/or blood is also unclear. There are very few studies investigating the direct effects of high CSF glucose levels on central nervous system (CNS) function, especially on learning and memory, since current diabetes models are not sufficiently developed to address such research questions. This article describes a method to chronically increase CSF glucose levels for 4 weeks by continuously infusing glucose into the lateral ventricle using osmotic minipumps in mice. The protocol was validated by measuring glucose levels in CSF. This protocol increased CSF glucose levels to ~328 mg/dL after infusion of a 50% glucose solution at a 0.25 &#181;L/h flow rate, compared to a CSF glucose concentration of ~56 mg/dL in mice that received artificial cerebrospinal fluid (aCSF). Furthermore, this protocol did not affect blood glucose levels. Therefore, this method can be used to determine the direct effects of high CSF glucose on brain function or a specific neural pathway independently of changes in blood glucose levels. Overall, the approach described here will facilitate the development of animal models for testing the role of high CSF glucose in mediating features of Alzheimer&#39;s disease and/or other neurodegenerative disorders associated with diabetes.

This article describes a detailed protocol to increase glucose concentration in the cerebrospinal fluid (CSF) of mice. This approach can be useful for studying the effects of high CSF glucose on neurodegeneration, cognition, and peripheral glucose metabolism in mice.

Diabetes increases the risk of cognitive decline and impairs brain function. Whether or not this relationship between high glucose and cognitive deficits ...

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Summary

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Chapters in this video

Mouse Round Spermatid Injection

An In Ovo Model for Testing Insulin-mimetic Compounds

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Modifying Levels of Maternal Dietary Folic Acid or Choline to Study the Impact of Deficiencies on Offspring Health Outcomes

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Osmotic Minipump Implantation for Increasing Glucose Concentration in Mouse Cerebrospinal Fluid