Name: OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery (Video) | JoVE Journal
Uploaded: 2021-05-09T14:00:00.000+00:00
Duration: 5 min 31 s
Description: Learn OP-IVM for fertility preservation in women requiring gynecological surgery. This method combines oocyte retrieval with procedures like laparoscopic ovarian drilling to improve ART outcomes.

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5 minutes

This work was supported by grants from the China National Key R&#38;D Program (no. 2017YFC1002000, 2018YFC1004001, 2019YFA0801400), the National Science Foundation of China (no. 81571386, 81730038), the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-001), and the Special Research Project of Chinese Capital Health Development (2018-2-4095).

NOTE: Studies related to the OP-IVM method have been approved by the institutional review board (IRB) of Peking University Third Hospital and the Ethics Committee of Peking University (2014S2004). A summary of OP-IVM is shown in Figure 1. The step-by-step procedure will be introduced in the following section.
1. Introduction of OP-IVM to appropriate patients
<ol>
	<li>Identify potential patients who may benefit from OP-IVM such as those described in steps 1.1.1-1.1.3.:
	<ol>
		<li>Polycystic ovarian syndrome (PCOS) patients with clomiphene resistance who need laparoscopic ovarian drilling surgery.</li>
		<li>Infertile patients who need benign gynecological surgeries, such as hysteroscopic myomectomy, polypectomy, transcervical resection of septum and laparoscopic tubal surgery, and oophorocystectomy, before ART treatment.</li>
		<li>Patients with cancer or hematological disease who are receiving chemoradiotherapy or radiotherapy.</li>
	</ol>
	</li>
</ol>
2. Informed consent
<ol>
	<li>Provide full information to patients, including why OP-IVM may be beneficial, its procedure, uses of IVM oocytes (IVF-ET or cryopreservation), estimated CPRs and LBRs, and possible complications. Ask patients to give informed consent by signing the consent form.</li>
</ol>
3. Prepare labels and IVM oocyte medium
<ol>
	<li>Print identification (ID) labels with the patient&#39;s name and dates for culture dishes and tubes.</li>
	<li>Add 0.5 mL of IVM&#160;medium supplemented with 0.75 IU/mL of follicle-stimulating hormone (FSH) and 0.75 IU/mL of luteinizing hormone (LH) to each well of a 4-well plate. Cover the medium with oil. 
	NOTE: Perform all these procedures in laminar flow clean benches.</li>
	<li>Prewarm the 4-well plate with IVM oocyte medium at 37 &#176;C in humidified air containing 5% CO2 and 5% O2 at least 6 h before use.</li>
</ol>
4. Administer anesthesia in an operating room
<ol>
	<li>Check the name of the patient before administering anesthesia.</li>
	<li>Intravenously anesthetize the patient by anesthesiologist.</li>
</ol>
5. Perform oocyte retrieval
<ol>
	<li>Tag the labels with name, date, and ID on the culture dishes and tubes.</li>
	<li>Place the patient in the bladder lithotomy position; constantly disinfect, drape, and scrub the vagina with warm saline.</li>
	<li>Place the ultrasound probe inside the vagina; scan and record the number of follicles in both&#160;ovaries. Find the location closest to the ovary as the puncture site, and avoid the intestine, bladder, and large blood vessels.</li>
	<li>Aspirate follicular fluid.
	<ol>
		<li>Wash the needle with&#160;pH-stable handling medium before puncturing (see Table of Materials).</li>
		<li>Inject the 19 G, single-lumen aspiration needle into the ovaries under the guidance of ultrasound.</li>
		<li>Puncture larger follicles with clear boundaries closest to the probe. At a low position, quickly inject the rinse solution supplemented with 25 U/mL of heparin. Aspirate follicular fluid with the needle under a pressure of 80-90 mmHg, rotating the needle slightly to aspirate as much follicular fluid as possible. Puncture other follicles from the near to far side on this plane. 
		NOTE: During aspiration, follicular fluid containing oocytes will flow into a sterile 10 mL test tube under negative pressure. Heparin can reduce the follicular fluid viscosity to facilitate the aspiration process.</li>
		<li>Remove the needle from the ovary (keep the needle in the vaginal wall). Adjust the direction of the ultrasound probe, and puncture the remaining follicles on other planes. Try to aspirate all the follicles with a diameter of ~5-9 mm. 
		&#8203;NOTE: Adjust the probe&#39;s position to keep it closest to the ovaries at all times. Press the vaginal fornix with appropriate force to reduce injury and bleeding.</li>
		<li>Pull out the needle after finishing in one ovary, wash the needle with the handling medium, and puncture the other side using the same method. 
		NOTE: Complete follicle aspiration within 25-30 min.</li>
	</ol>
	</li>
	<li>Transfer the follicular fluid from the operation room to the IVF laboratory within a few minutes after confirming the patient&#39;s name and ID. 
	NOTE: Transfer aspirated follicular fluid to the IVF laboratory bench as soon as possible to prevent coagulation.</li>
	<li>Detect active hemorrhage in the pelvic cavity with B-mode ultrasound after puncturing all the follicles. Insert a speculum, and point the tip at the posterior fornix to detect active bleeding at the puncture site. 
	&#8203;NOTE: No active bleeding should be observed at the vaginal puncture site if the ovary position is normal&#160;and oocyte retrieval is performed carefully. For light bleeding that continues even after compression, keep a sterile gauze compress in the vagina for 2-4 h. Control excessive bleeding from small arteries using clamps with vascular forceps for 2-4 h. Bleeding in the pelvic cavity or ovary, which seldom happens, should be controlled by electrocoagulation by laparoscopic surgery.</li>
</ol>
6. Gynecological surgery
<ol>
	<li>Based on the need and condition of the patient, perform appropriate gynecological surgery after oocyte retrieval.
	<ol>
		<li>Perform laparoscopic ovarian drilling surgery for polycystic ovarian syndrome (PCOS) patients with clomiphene resistance.</li>
		<li>Perform benign gynecological surgery for infertile patients before ART treatment, such as hysteroscopic myomectomy, polypectomy, transcervical resection of septum and laparoscopic tubal surgery, and oophorocystectomy.</li>
		<li>Perform ovariectomy for fertility cryopreservation of patients with cancer or hematological disease who need to receive chemoradiotherapy. 
		&#8203;NOTE: These gynecological surgeries are basic and standardized clinical operations. Operation guidelines in various countries and hospitals should be relatively similar.</li>
	</ol>
	</li>
</ol>
7. Perform IVM
NOTE: Perform the whole process of IVM on a 37 &#176;C homothermal flat.
<ol>
	<li>Filter the aspirated follicle fluid with a 70 &#181;m nylon cell strainer. Repeatedly rinse the culture tube and strainer with pre-warmed pH-stable handling medium. Ensure all the immature COCs are completely transferred to the culture dish. Collect the filtered fluid , rinse, and culture in a 100 x 15 mm Petri dish.</li>
	<li>Examine the COCs (Figure 2) under the stereoscope with 40x magnification. Quickly transfer the immatures into a pre-warmed IVM oocyte medium. 
	&#8203;NOTE: Choose an appropriate magnification depending on the operator&#39;s habit.</li>
	<li>Record the number of cultured immature COCs.</li>
	<li>Inform the patient about the number of cultured COCs. Discuss the collection of semen with the patients and their partners. Perform sperm extraction according to the normal procedure.</li>
	<li>Culture the immature COCs at 37 &#176;C in humidified air containing 5% CO2 and 5% O2 for 28-32 h.</li>
	<li>Assess the oocyte maturity.
	<ol>
		<li>Denude the cumulus cells by repeated pipetting using a glass Pasteur pipette under the stereoscope with 40x magnification. Examine the extrusion of the polar body (PB) to identify the developmental stage of the oocytes. See Figure 2 for representative images of oocytes in COCs, metaphase II (MII), metaphase I (MI), and germinal vesicles (GVs) with clear morphological characteristics. 
		NOTE: Use a vertical flame (e.g., of a Bunsen burner) to adjust the diameter of the glass Pasteur pipette to the size of an oocyte.</li>
		<li>Count MII oocytes and record the number.</li>
		<li>Choose mature oocytes for IVF or vitrification. 
		&#8203;NOTE: Collect sperm on the day the oocytes mature for IVF, but not for oocyte vitrification.</li>
		<li>Culture the immature oocytes in GV and MI stage in the original IVM culture medium with cumulus cells for another 10-14 h. Repeat steps 7.6.2-7.6.3.</li>
	</ol>
	</li>
</ol>
8. Perform ICSI or oocyte vitrification
<ol>
	<li>Follow the standard procedure of the reproductive center8.</li>
</ol>
9. Culture embryos and perform embryo cryopreservation
<ol>
	<li>Follow the standard procedure of the reproductive center8.</li>
</ol>

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<li>Katayama, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasound-guided+transvaginal+needle+aspiration+of+follicles+for+in+vitro+fertilization%5BTitle%5D)+AND+%22Obstetrics+and+Gynecology%22%5BJournal%5D)">Ultrasound-guided transvaginal needle aspiration of follicles for in vitro fertilization.</a> Obstetrics and Gynecology. 72 (2), 271-274 (1988).</li>
<li>Dellenbach, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transvaginal+sonographically+controlled+follicle+puncture+for+oocyte+retrieval%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Transvaginal sonographically controlled follicle puncture for oocyte retrieval.</a> Fertility and Sterility. 44 (5), 656-662 (1985).</li>
<li>Hirata, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concomitant+ovarian+drilling+and+oocyte+retrieval+by+laparoendoscopic+single-site+surgery+led+to+live+birth+using+in+vitro+maturation+of+oocyte+and+transfer+of+frozen-thawed+blastocyst+in+woman+with+polycystic+ovary+syndrome%5BTitle%5D)+AND+%22Journal+of+Obstetrics+and+Gynaecology+Research%22%5BJournal%5D)">Concomitant ovarian drilling and oocyte retrieval by laparoendoscopic single-site surgery led to live birth using in vitro maturation of oocyte and transfer of frozen-thawed blastocyst in woman with polycystic ovary syndrome.</a> Journal of Obstetrics and Gynaecology Research. 40 (5), 1431-1435 (2014).</li>
<li>Deutinger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Follicular+aspiration+for+in+vitro+fertilization%3A+sonographically+guided+transvaginal+versus+laparoscopic+approach%5BTitle%5D)+AND+%22European+Journal+of+Obstetrics%2C+Gynecology%2C+and+Reproductive+Biology%22%5BJournal%5D)">Follicular aspiration for in vitro fertilization: sonographically guided transvaginal versus laparoscopic approach.</a> European Journal of Obstetrics, Gynecology, and Reproductive Biology. 26 (2), 127-133 (1987).</li>
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<li>Nagy, Z. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pregnancy+and+birth+after+intracytoplasmic+sperm+injection+of+in+vitro+matured+germinal-vesicle+stage+oocytes%3A+case+report%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report.</a> Fertility and Sterility. 65 (5), 1047-1050 (1996).</li>
<li>Söderström-Anttila, V., Mäkinen, S., Tuuri, T., Suikkari, A. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Favourable+pregnancy+results+with+insemination+of+in+vitro+matured+oocytes+from+unstimulated+patients%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients.</a> Human Reproduction. 20 (6), 1534-1540 (2005).</li>
<li>Hwang, J. L., Lin, Y. H., Tsai, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+maturation+and+fertilization+of+immature+oocytes%3A+a+comparative+study+of+fertilization+techniques%5BTitle%5D)+AND+%22Journal+of+Assisted+Reproduction+and+Genetics%22%5BJournal%5D)">In vitro maturation and fertilization of immature oocytes: a comparative study of fertilization techniques.</a> Journal of Assisted Reproduction and Genetics. 17 (1), 39-43 (2000).</li>
<li>Walls, M., Junk, S., Ryan, J. P., Hart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IVF+versus+ICSI+for+the+fertilization+of+in-vitro+matured+human+oocytes%5BTitle%5D)+AND+%22Reproductive+Biomedicine+Online%22%5BJournal%5D)">IVF versus ICSI for the fertilization of in-vitro matured human oocytes.</a> Reproductive Biomedicine Online. 25 (6), 603-607 (2012).</li>
<li>Park, J. H., Jee, B. C., Kim, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+normal+and+abnormal+fertilization+of+in+vitro-matured+human+oocyte+according+to+insemination+method%5BTitle%5D)+AND+%22Journal+of+Obstetrics+and+Gynaecology+Research%22%5BJournal%5D)">Comparison of normal and abnormal fertilization of in vitro-matured human oocyte according to insemination method.</a> Journal of Obstetrics and Gynaecology Research. 42 (4), 417-421 (2016).</li>
<li>Ho, V. N. A., Pham, T. D., Le, A. H., Ho, T. M., Vuong, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+birth+rate+after+human+chorionic+gonadotropin+priming+in+vitro+maturation+in+women+with+polycystic+ovary+syndrome%5BTitle%5D)+AND+%22Journal+of+Ovarian+Research%22%5BJournal%5D)">Live birth rate after human chorionic gonadotropin priming in vitro maturation in women with polycystic ovary syndrome.</a> Journal of Ovarian Research. 11 (1), 70(2018).</li>
<li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+hCG+priming+on+embryonic+development+of+immature+oocytes+collected+from+unstimulated+women+with+polycystic+ovarian+syndrome%5BTitle%5D)+AND+%22Reproductive+Biology+and+Endocrinology%22%5BJournal%5D)">Effect of hCG priming on embryonic development of immature oocytes collected from unstimulated women with polycystic ovarian syndrome.</a> Reproductive Biology and Endocrinology. 10, 40(2012).</li>
<li>Tannus, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictive+factors+for+live+birth+after+in+vitro+maturation+of+oocytes+in+women+with+polycystic+ovary+syndrome%5BTitle%5D)+AND+%22Archives+of+Gynecology+and+Obstetrics%22%5BJournal%5D)">Predictive factors for live birth after in vitro maturation of oocytes in women with polycystic ovary syndrome.</a> Archives of Gynecology and Obstetrics. 297 (1), 199-204 (2018).</li>
<li>Cohen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+pregnancy+and+live+birth+rates+after+vitrification+of+in+vitro+matured+oocytes%5BTitle%5D)+AND+%22Journal+of+Assisted+Reproduction+and+Genetics%22%5BJournal%5D)">Decreased pregnancy and live birth rates after vitrification of in vitro matured oocytes.</a> Journal of Assisted Reproduction and Genetics. 35 (9), 1683-1689 (2018).</li>
<li>Yazdanpanah, F., Khalili, M. A., Eftekhar, M., Karimi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+vitrification+on+maturation+and+viability+capacities+of+immature+human+oocytes%5BTitle%5D)+AND+%22Archives+of+Gynecology+and+Obstetrics%22%5BJournal%5D)">The effect of vitrification on maturation and viability capacities of immature human oocytes.</a> Archives of Gynecology and Obstetrics. 288 (2), 439-444 (2013).</li>
</ol>

The authors have nothing to disclose.

The OP-IVM method described in this article extends to conventional IVM applications and combines IVM after oocyte retrieval with routine gynecological surgery. Oocytes that would have been lost in the gynecological surgery can now be used for IVF-ET or FP without additional surgical risks. OP-IVM was first used to retrieve oocytes before ovarian drilling in PCOS patients. Its application soon expanded to infertile patients who need benign gynecological surgery and cancer or hematological disease patients who need chemor...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61647">OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery</a>. The Protocol and Representative Results sections were&#160;updated.
Step 3.2 of the Protocol was updated from:
Add 0.5 mL of IVM&#160;medium supplemented with 0.075 IU/mL of follicle-stimulating hormone (FSH) and 0.075 IU/mL of luteinizing hormone (LH) to each well of a 4-well plate. Cover the medium with oil.
to:
Add 0.5 mL of IVM&#160;medium supplemented with 0.75 IU/mL of follicle-stimulating hormone (FSH) and 0.75 IU/mL of luteinizing hormone (LH) to each well of a 4-well plate. Cover the medium with oil.
The legend for Figure 2 was updated from:
Figure 2:&#160;Morphological characteristics of representative images of oocytes. (A) COCs, (B) MII, (C) MI, (D) GV stage. Abbreviations: COCs = cumulus-oocyte complexes; GV = germinal vesicle; MI = metaphase I; MII = metaphase II.
to:
Figure 2:&#160;Morphological characteristics of representative images of oocytes. (A) COCs, (B) GV, (C) MI, (D) MII&#160;stage. Abbreviations: COCs = cumulus-oocyte complexes; GV = germinal vesicle; MI = metaphase I; MII = metaphase II.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61647">OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery</a>. The Protocol and Representative Results sections were&#160;updated.

Erratum: OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

IVM is an ART in which human immature oocytes are cultured in vitro to maturation for IVF-ET or FP. In IVM, ovulation induction medications are not used, thus reducing pain, financial burden, and complications such as ovarian hyperstimulation syndrome (OHSS)1,2. In addition, IVM is particularly suitable for the FP of cancer patients and the infertility treatment of hormone-sensitive patients who may be unable to or have no time to receive ovulation induction therapy3. Therefore, although the number of oocytes retrieved, clinical pregnancy rate (CPR), and live birth rate (LBR) are lower than those of IVF4,5, IVM has its own unique advantages.
Infertile patients with endometrial lesions, hydrosalpinx, or ovarian cysts usually have gynecological surgery before ART treatment, and their oocytes are usually immature. OP-IVM uses guided transvaginal ultrasound to retrieve the immature oocytes and grow them in vitro until maturation for IVF-ET or FP. OP-IVM combines IVM after oocyte retrieval and gynecological surgery, thereby reducing complications that are common in&#160;controlled ovarian hyperstimulation cycles and saving time and money. For fertile patients, OP-IVM could serve as a &#34;fertility insurance&#34; while undergoing routine gynecological surgery.
Furthermore, damages caused by gynecological surgeries, such as electrocautery6,7 and ovarian tumor resection, could be reduced through oocyte retrieval before gynecological surgery. Therefore, compared with routine gynecological surgery, OP-IVM could reduce the number of operations during infertility treatment and prevent the loss of functional oocytes during ovarian surgery.
A previous study has shown that the additional procedure of oocyte retrieval would neither increase surgical complications and adverse pregnancy outcomes, nor prolong the hospital stay8. Some patients have given live birth through OP-IVM8, indicating the feasibility of this method. This paper describes characteristics of patients who may benefit from OP-IVM as well as procedures and critical points of OP-IVM and discusses the evaluation of human oocyte maturity.

<table><tbody><tr><td>19 G single-lumen aspiration needles</td><td>Cook, Australia</td><td>K-OPS-7035-REH-ET</td><td></td></tr><tr><td>4-well plate</td><td>Corning</td><td></td><td></td></tr><tr><td>70 &amp;mu;m nylon cell strainer</td><td>Falcon, USA</td><td>352350</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo</td><td></td><td></td></tr><tr><td>Culture oil</td><td>Vitrolife, Sweden</td><td>10029,OVOIL&amp;nbsp;</td><td>Step 3.2.</td></tr><tr><td>FSH &amp;amp; LH</td><td>Ferring Reproductive Health, Germany</td><td>MENOPUR&amp;reg;</td><td></td></tr><tr><td>Glass Pasteur pipette</td><td>Hilgenberg GmbH, Germany</td><td>3154102-26</td><td></td></tr><tr><td>G-MOPS medium</td><td></td><td></td><td>pH-stable handling medium for washing the needle before puncturing</td></tr><tr><td>IVM medium</td><td>Origio, Denmark</td><td>ART-1600-B</td><td></td></tr><tr><td>Laminar Flow Clean Benches</td><td>ESCO</td><td></td><td></td></tr><tr><td>Petri dish</td><td>Thermo Fisher Scientific, Denmark</td><td>263991</td><td></td></tr><tr><td>pH stable handing media designed to support the handling and manipulation of oocytes and embryos outside the incubator</td><td>Vitrolife, Sweden</td><td>10130, G-MOPS PLUS</td><td>Step 7.1.</td></tr><tr><td>Rinse solution</td><td>Cook, Australia</td><td>K-SIFB-100</td><td></td></tr><tr><td>Stereoscope</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

op-ivm: combining in vitro maturation after oocyte retrieval with gynecological surgery

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte retrieval with routine gynecological surgery. OP-IVM is suitable for patients undergoing benign gynecological surgery who have the need for fertility preservation (FP) or infertility treatments such as in vitro fertilization and embryo transfer (IVF-ET). In the operating room, patients undergoing benign gynecological surgery are first anesthetized and receive ultrasound-guided immature follicle aspiration (IMFA) treatment. As the subsequent gynecological surgery is performed, the cumulus-oocyte complexes (COCs) are examined, and the immature COCs are transferred into the IVM medium and cultured for 28-32 hours in the IVF laboratory. After assessment, mature oocytes in the MII stage will be selected and cryopreserved in liquid nitrogen for FP or fertilized by intracytoplasmic sperm injection (ICSI) for IVF-ET. By combining IVM with gynecological surgery, immature oocytes that would have been discarded can be saved and used for assisted reproductive technology (ART). The procedure, significance and critical aspects of OP-IVM are described in this article.

Until December 2019, OP-IVM was used for fertility preservation of 274 patients. Embryological and reproductive outcomes of 158 patients between 2014 to 2016 were published in a previous paper8. The following example discusses the procedure followed for a PCOS patient receiving OP-IVM in 2016. The patient is a 28-year-old diagnosed with primary infertility, left adnexal cyst, and PCOS. She received laparoscopic cystectomy and OP-IVM on September 28th, 20...

In vitro maturation (IVM) before gynecological operation (OP-IVM) combines IVM following oocyte retrieval with routine gynecological surgery and serves as an extension of conventional IVM applications for fertility preservation.

The use of in vitro maturation (IVM) before gynecological operation (OP-IVM) is an extension of conventional IVM that combines IVM following oocyte ...

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Research

JoVE Journal

Medicine

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage.

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women. Appropriate clinical and laboratory strategies ensure maximum efficacy, efficiency, and safety of fertility preservation treatments.

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

Fertility Preservation in Patients with Severe Ovarian Dysfunction

A dedicated team can offer Mayer-Rokitansky-Kuster-Hauser patients the option to perform controlled ovarian stimulation and oocyte cryopreservation at the time of laparoscopic vaginoplasty.

Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome

Here, we present an optimized method for promptly removing a portion of cumulus-oocyte complexes following egg retrieval to expedite the IVF observation process, reducing the time required for granulosa cell removal, minimizing oocyte exposure to external elements, and does not hinder embryo development, ultimately improving the efficiency of IVF laboratory procedures.

The Immediate Partial Removal of Cumulus-Oocyte Complexes: A Refined Approach for Rapid Observation of In Vitro Fertilization

This protocol outlines standardized methods to isolate and process follicular fluid, somatic cells, and immature oocytes from IVF procedures, enabling high-quality molecular and cellular analyses. These approaches support translational research into fertility, reproductive aging, and ovarian dysfunction using materials typically discarded in clinical care.

Collection of Human Follicular Fluid, Follicle Somatic Cells, and Immature Oocytes from Individuals Undergoing In Vitro Fertilization

This protocol describes procedures for in vitro maturation of mouse oocytes, in vitro fertilization, and subsequent embryo culture to the blastocyst stage.

Mouse Oocyte In Vitro Maturation, Fertilization, and Culture of Preimplantation Embryos

Here we describe how to set up a small microsurgical practice station and a simple and inexpensive method for the training of microsurgery with non-living animal models.



Step By Step: Microsurgical training method combining two nonliving animal models

Learn OP-IVM for fertility preservation in women requiring gynecological surgery. This method combines oocyte retrieval with procedures like laparoscopic ovarian drilling to improve ART outcomes.

OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery

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