<meta charset="utf8"></head><body>ICSI-ET 小鼠模型研究对葡萄糖代谢的长期影响

<ol>
	<li>Palermo, G., Joris, H., Devroey, P., Van Steirteghem, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnancies+after+intracytoplasmic+injection+of+single+spermatozoon+into+an+oocyte.">Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte.</a> Lancet. 340 (8810), 17-18 (1992).</li><li>Flurkey, K. M., Currer, J., Harrison, D. E., Fox, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vol.+III+Mouse+models+in+aging+research.+Ch.+20.">Vol. III Mouse models in aging research. Ch. 20.</a> The mouse in biomedical research (second edition). , 637-672 (2007).</li><li>Chen, M., Heilbronn, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+health+outcomes+of+human+offspring+conceived+by+assisted+reproductive+technologies+(art).">The health outcomes of human offspring conceived by assisted reproductive technologies (art).</a> J Dev Orig Health Dis. 8 (4), 388-402 (2017).</li><li>Heber, M. F., Ptak, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+assisted+reproduction+technologies+on+metabolic+health+and+diseasedagger.">The effects of assisted reproduction technologies on metabolic health and diseasedagger.</a> Biol Reprod. 104 (4), 734-744 (2021).</li><li>Ma, R. C. W., Ng, N. Y. H., Cheung, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assisted+reproduction+technology+and+long-term+cardiometabolic+health+in+the+offspring.">Assisted reproduction technology and long-term cardiometabolic health in the offspring.</a> PLoS Med. 18 (9), e1003724 (2021).</li><li>De Waal, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cumulative+effect+of+assisted+reproduction+procedures+on+placental+development+and+epigenetic+perturbations+in+a+mouse+model.">The cumulative effect of assisted reproduction procedures on placental development and epigenetic perturbations in a mouse model.</a> Hum Mol Genet. 24 (24), 6975-6985 (2015).</li><li>Carlin, J., George, R., Reyes, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methyl+donor+supplementation+blocks+the+adverse+effects+of+maternal+high+fat+diet+on+offspring+physiology.">Methyl donor supplementation blocks the adverse effects of maternal high fat diet on offspring physiology.</a> PLoS One. 8 (5), e63549 (2013).</li><li>Hiraoka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezo-icsi+for+human+oocytes.">Piezo-icsi for human oocytes.</a> J Vis Exp. (170), e60224 (2021).</li><li>Sullivan-Pyke, C. S., Senapati, S., Mainigi, M. A., Barnhart, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fertilization+and+adverse+obstetric+and+perinatal+outcomes.">In vitro fertilization and adverse obstetric and perinatal outcomes.</a> Semin Perinatol. 41 (6), 345-353 (2017).</li><li>Luke, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+risk+of+birth+defects+with+conception+by+art.">The risk of birth defects with conception by art.</a> Hum Reprod. 36 (1), 116-129 (2021).</li><li>Norrman, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+disease,+obesity,+and+type+2+diabetes+in+children+born+after+assisted+reproductive+technology:+A+population-based+cohort+study.">Cardiovascular disease, obesity, and type 2 diabetes in children born after assisted reproductive technology: A population-based cohort study.</a> PLoS Med. 18 (9), e1003723 (2021).</li><li>Goldsmith, S., Mcintyre, S., Badawi, N., Hansen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+palsy+after+assisted+reproductive+technology:+A+cohort+study.">Cerebral palsy after assisted reproductive technology: A cohort study.</a> Dev Med Child Neurol. 60 (1), 73-80 (2018).</li><li>Hansen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intellectual+disability+in+children+conceived+using+assisted+reproductive+technology.">Intellectual disability in children conceived using assisted reproductive technology.</a> Pediatrics. 142 (6), e20181269 (2018).</li><li>Chang, R. C., Wang, H., Bedi, Y., Golding, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconception+paternal+alcohol+exposure+exerts+sex-specific+effects+on+offspring+growth+and+long-term+metabolic+programming.">Preconception paternal alcohol exposure exerts sex-specific effects on offspring growth and long-term metabolic programming.</a> Epigenetics Chromatin. 12 (1), 9 (2019).</li><li>Grasemann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parental+diabetes:+The+akita+mouse+as+a+model+of+the+effects+of+maternal+and+paternal+hyperglycemia+in+wildtype+offspring.">Parental diabetes: The akita mouse as a model of the effects of maternal and paternal hyperglycemia in wildtype offspring.</a> PLoS One. 7 (11), e50210 (2012).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 <img alt="Equation 1" src="/files/ftp_upload/66212/66212eq01.jpg" style="margin: auto;" /> 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&#160;</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&#39;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

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.

<meta charset="utf8"></head><body>作者聚焦：探索卵胞浆内单精子注射对后代的长期健康影响

监测胞浆内单精子注射后小鼠后代的血糖

We're looking into whether the techniques used in ICSI might cause metabolic disorder in offspring. It's really important because it ensures the safety and development of assisted reproductive technology. Our research provide a solid foundation for future study enabling researchers to explore how sperm impact on embryo growth and the offspring health over time.Our team will focus on cardiovascular health and the immune system outcomes in ICSI offspring aiming to provide a comprehensive understanding of their long-term health.

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

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 ...

monitoring-blood-glucose-mouse-offspring-after-intracytoplasmic-sperm

Research

JoVE Journal

Developmental Biology

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

342 次观看.  Shanghai-MOST Key Laboratory of Health and Disease Genomics. 我们正在研究 ICSI 中使用的技术是否会导致后代代谢紊乱。这真的很重要，因为它确保了辅助生殖技术的安全和发展。我们的研究为未来的研究奠定了坚实的基础，使研究人员能够探索精子如何随着时间的推移对胚胎生长和后代健康产生影响。我们的团队将专注于 ICSI 后代的心血管健康和免疫系统结果，旨在全面了解他们的长期健康状况。

视频: 作者聚焦：探索卵胞浆内单精子注射对后代的长期健康影响

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.

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.

科研

发育生物学

Intracytoplasmic Sperm Injection (ICSI) and Metabolic Assessments in Mice 

To begin, place the experimental female mouse on a working platform. Intraperitoneally, administer 7.5 international units of pregnant mare serum gonadotropin around 8:00 PM on the first day. After 48 hours, intraperitoneally inject 7.5 international units of human chorionic gonadotropin.Divide a cell culture dish lid into upper and lower halves. In the upper half, place four to six droplets of five microliter each of PVP for sperm placement. Add 8 to 10 droplets of five microliter each of M2 medium in the lower half for the ICSI procedure.Cover the PVP and M2 medium droplets with approximately three milliliters of mineral oil. After isolating epididymal cauda from the euthanized male mouse, gently blot it on sterilized gauze. Cut open the cauda epididymis and gently squeeze, using forceps to release the sperm, collecting them in pre-equilibrated human tubal fluid tubes.Rinse the cauda epididymis in the pre-warmed human tubal fluid medium. Place the tube uncovered at 37 degrees Celsius and 5%carbon dioxide incubator for sperm capacitation. On the fourth day after injecting human chorionic gonadotropin, place the euthanized female mouse under a dissecting microscope.Using a 25-gauge needle, tear open the ampulla of the oviduct to release numerous cumulus oocyte complexes. After capacitation, sonicate 100 microliters of the sperm for five seconds to separate the heads from the tails. Fill the micromanipulation needle with the operating fluid, ensuring the filled portion measures 0.5 centimeters inside the needle.Install the needle on the right arm of the micromanipulator and secure it by tightening the holding cap. Then install a flat-tipped micromanipulation holding pipette on the left arm of the micromanipulator. Position the injection needle and the holding pipette in the microscope's field of view.Under a polarization microscope, determine the position of the metaphase spindle within the oocyte to ensure that the spindle apparatus is not on the injection side. Upon contact of the injection needle with the zona pellucida, activate Piezo pulses with an intensity of five and frequency of one, creating a perforation in the zona pellucida and allowing the injection needle to pass through. Using Piezo pulses of intensity one and frequency one, create a small pore in the plasma membrane, ensuring the sperm head is injected into the ooplasm.Using atraumatic forceps, carefully exteriorize the ovaries, oviducts, and uterus from the anesthetized pseudo-pregnant female mouse. Make a small upward-angled incision using the tip of a syringe needle on the uterine wall below the uterotubal junction, avoiding blood vessels. Gently insert the oviduct two to three millimeters through the small hole.Using the ICSI method, a fertilization rate of 89.57%was achieved in mice, with 87.38%of zygotes advancing to the 2-cell embryo stage within 24 hours post ICSI. The birth rate of live pups post embryo transfer was observed at 42.5%There was no significant fluctuation in random blood glucose levels between mice born naturally and ICSI mice. However, male ICSI mice consistently exhibited elevated fasting blood glucose levels, suggesting impaired glucose homeostasis.In glucose tolerance tests, significant differences in blood glucose levels were noted between male ICSI and control mice, while no difference was observed in female mice. In body weight tracking, both male and female ICSI mice exhibited an overweight phenotype compared to controls.