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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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 chemoradiotherapy. Because of the potential risk of metastasis, OP-IVM is not suitable or recommended for patients with malignant tumors. Based on OP-IVM, a new Chinese mode of egg bank for the FP of infertile patients and cancer patients has been established at this institution.
For OP-IVM, both the urgency of gynecological surgery and the potential of obtaining a higher number of oocytes should be considered. In general, IVM outcome is positively correlated with the number of immature oocytes obtained during oocyte retrieval9. Dominant follicles may inhibit the growth of surrounding smaller follicles and cause atresia10, resulting in a reduced number of retrieved immature oocytes. Therefore, if the gynecological surgery is not urgent, the best window for OP-IVM is when a greater number of small follicles with few dominant follicles are observed by B-mode ultrasonography. In addition, anti-Mullerian hormone (AMH) can be used as a parameter to predict ovarian reserve as the numbers of retrieved oocytes are significantly positively correlated with AMH11,12. In cases where gynecological operation has been prioritized, as many immature oocytes should be retrieved as much as possible. In emergency operations, oocyte retrieval and IVM should be carefully evaluated based on the patient&#39;s condition and will.
IMFA under transvaginal ultrasound guidance is a method used to retrieve oocytes that can reduce damage to ovarian function13,14. Laparoscopic ovarian puncture to aspirate immature oocytes is also used in some studies15. However, compared with laparoscopy, IMFA under transvaginal ultrasound takes up less time, is less invasive, is easier to perform16,17, and can achieve more targeted follicle aspiration. Therefore, to minimize the impact on ovarian reserve and increase aspiration accuracy, IMFA under transvaginal ultrasound guidance is recommended as a better method for oocyte retrieval in OP-IVM. However, ICSI is recommended for fertilization. Previous studies have shown that the zona pellucida will harden after being cultured in vitro for long periods, resulting in a reduced rate of successful fertilization18. ICSI has been shown to effectively improve the fertilization rate of IVM oocytes in natural cycles19,20. Although recent studies have shown similar fertilization rates for ICSI and IVF21,22, ICSI holds the advantage of using natural cycles instead of ovarian stimulation. Collected embryos are frozen because of unprepared endometrium, and embryo transfer can be performed later according to the standard procedure at each center.
The purpose of ART is to treat infertility and deliver healthy babies. Previous research about the reproductive outcomes of OP-IVM showed that the numbers of retrieved oocytes and oocyte maturation rates are comparable with those observed for conventional IVM cycles using FSH and hCG. However, OP-IVM has a lower CPR and a lower LBR than conventional IVM8,23,24,25. This may be explained by constrained oocyte maturation due to the poor development potential of immature oocytes in natural cycles. However, hormone stimulation is not an option for some patients who are unable to or who do not have the time to receive the treatment. In addition, vitrification may damage the oocytes&#39; developmental potential, resulting in low CPRs and LBRs26,27.
Moreover, gonadotropins may enlarge the ovaries and increase the risk of bleeding during oocyte retrieval, adversely affecting the exposure of operative fields in laparoscopy. The LBR of the OP-IVM method is relatively low. More studies are needed to examine the best operative timing and modify the IVM culture system to improve OP-IVM outcomes. Overall, OP-IVM combines IVM with gynecological surgery so that immature oocytes that would have been damaged or discarded can be saved and used for ART. OP-IVM has the advantages of avoiding complications caused by ovarian stimulating medications and reducing the number of operations for infertility treatment. Therefore, OP-IVM should be considered as a potentially valuable treatment method for certain patients and studied more in depth to better understand its effects and outcomes.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#956;m nylon cell strainer</td>
			<td>Falcon, USA</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture oil</td>
			<td>Vitrolife, Sweden</td>
			<td>10029,OVOIL&#160;</td>
			<td>Step 3.2.</td>
		</tr>
		<tr>
			<td>FSH &#38; LH</td>
			<td>Ferring Reproductive Health, Germany</td>
			<td>MENOPUR&#174;</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

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, 2016; 27 immature COCs were obtained. After a 28 h culture, 7 oocytes in the MII stage were selected and fertilized by ICSI. The number of good-quality 2 pro-nuclei (2PNs) on day 1 and embryos on day 3, and day 5 were 6, 6, and 2, respectively. The blastocysts obtained on day 5 (October 4th, 2016) were frozen. On February 14th, 2017, one blastocyst was thawed. This thawed blastula was alive and transferred into the uterus on February 14th, 2017. Results of a blood human chorionic gonadotropin (hCG) test were positive, and ultrasonography showed intrauterine early pregnancy. The patient delivered a healthy girl by cesarean section at 40 weeks on November 2nd, 2017.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61647/61647fig01.jpg" /> 
Figure 1:&#160;OP-IVM flow chart. Before gynecological surgery, under transvaginal ultrasound guidance, follicle fluid is aspirated through IMFA. The obtained follicle fluid is transferred into the IVF laboratory, filtered, and rinsed. Immature COCs are transferred into a prewarmed IVM culture solution and cultured for 28-32 h. Mature oocytes with PB are used for cryopreservation or IVF-ET. Abbreviations: IMFA = immature follicle aspiration; IVF = in vitro fertilization; IVF-ET = IVF and embryo transfer; COCs = cumulus-oocyte complexes; IVM = in vitro maturation; OP-IVM = IVM before a gynecological operation; GV = germinal vesicle; MI = metaphase I; ICSI = intracytoplasmic sperm injection; PB = polar body; PN = pro-nucleus; D1 = day 1. <a href="https://www.jove.com/files/ftp_upload/61647/61647fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61647/61647fig2v2.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/61647/61647fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about OP-IVM: Combining In vitro Maturation after Oocyte Retrieval with Gynecological Surgery at JoVE.com

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

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 ...

op-ivm-combining-vitro-maturation-after-oocyte-retrieval-with

Nanyang Technological University

Research

JoVE Journal

Medicine

3.8K Views.  Peking University Third Hospital. 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.

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

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

The learning of microsurgical techniques and the maintenance of microsurgical skills have been traditionally based on the use of living animals, mainly laboratory rats. This method although extremely valuable can be economically demanding both for the surgeon and the sponsoring institution; it also requires special training facilities that may not always be available or accessible. Furthermore ethical concerns can limit the use of living animals for training purposes. Alternative training methods, such as inert tubes and gloves have not gained popularity among surgeons since they do not offer an experience similar to that of a clinical situation. Non-living animal models include the use of chicken thighs and wings; they offer a practice experience that resembles a clinical situation to a considerable extent. This type of training is relatively cheap and easily available. The microscope and instruments required can be acquired over the internet, and the chicken pieces can be bought at the local supermarket.
This approach allows a motivated trainee to rehearse different types of surgical techniques several times at a reasonable expense, helping to develop or maintain his surgical expertise if more complex facilities are not available. On the current manuscript we describe how to setup a small practice station, how to dissect the specimens, and how to practice both with the chicken thighs and with the chicken wings in a progressive fashion. This approach takes advantage on the versatility of the chicken thigh model and the small size of the chicken wing Brachial artery.

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.

The learning of microsurgical techniques and the maintenance of microsurgical skills have been traditionally based on the use of living animals, mainly ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

A Case Series of Successful Abdominal Closure Utilizing a Novel Technique Combining a Mechanical Closure System with a Biologic Xenograft that Accelerates Wound Healing

In the acute setting, once intra-abdominal injuries have been addressed, the next great hurdle is restoring a functional and intact abdominal compartment. The short and long-term consequences of living with a chronically open abdominal compartment include pulmonary, musculoskeletal, gastrointestinal, and emotional disability. The closure of catastrophic open abdomens presents a challenge to the surgeon. We present a technique utilizing a mechanical abdominal closure device in conjunction with biologic xenograft in closing complex open abdomens. This technique offers another option for definitive fascial closure and accelerated wound healing in this difficult patient population. The dynamic tissue system (DTS) is installed after control of original intraabdominal pathology. A porcine urinary bladder matrix (PUBM) is then placed in the subcutaneous space once fascial closure is achieved. Overall, primary myofascial closure was achieved in 100% of patients at a mean of 9.36 days.

Closure of catastrophic open abdominal wounds presents a challenge to the surgeon. We present a surgical technique utilizing a combination of mechanical and biologic xenograft closure systems in closing complex open abdominal wounds. This technique offers another option to the surgeon for definitive fascial closure and accelerated wound healing.

In the acute setting, once intra-abdominal injuries have been addressed, the next great hurdle is restoring a functional and intact abdominal compartment. ...

Intraoperative Ultrasound in Spinal Surgery

Since the 1980s, there have been several reports for the use of intraoperative ultrasound as a useful adjunct in spinal surgery. However, with the advent of newer cutting-edge imaging modalities, the use of intraoperative ultrasound in spine surgery has largely fallen out of favor. Despite this, intraoperative ultrasound continues to provide several advantages over other intraoperative techniques such as magnetic resonance imaging and computed tomography including being more cost-effective, efficient, and easy to operate and interpret. Additionally, it remains the only method for the real-time visualization of soft tissue and pathologies. This paper focuses on the advantages of using intraoperative ultrasound, especially in cases of intradural lesions and lesions ventral to the thecal sac when approaching posteriorly.

Here, we present a protocol on the use of intraoperative ultrasound in spinal surgery, particularly in cases of intradural lesions and lesions in the ventral spinal canal when using a posterior approach.

Since the 1980s, there have been several reports for the use of intraoperative ultrasound as a useful adjunct in spinal surgery. However, with the advent ...

Evaluation of Patients' Posture and Gait Profile After Lumbar Fusion Surgery by Video Rasterstereography and Treadmill Gait Analysis

This protocol provides guidance on how to perform high resolution video rasterstereography and treadmill gait analysis on patients after lumbar fusion surgery to obtain results about altered variables of gait and posture. These observed changes can then be correlated with the patient-reported outcome measure of pain relief. The rasterstereographic device projects lines of parallel light onto the surface of the tested subject&#39;s back. The deformation of these lines is recognized by the device. From these data, a special software then generates a 3-D profile based on the principle of triangulation. With an inaccuracy of only 0.2 mm it can measure changes in posture at very high precision. Gait and stance parameters are recorded using a treadmill equipped with an electric sensor mat that contains 10,200 miniature force sensors in the registering zone under the belt. Initial walking speed on the treadmill is 0.5 km/h. Speed is then gradually increased by increments of 0.1 km/h until each subject reaches his or her individual maximum well tolerable walking speed. At this speed, parameters are recorded during a 20 s measurement interval. Subjects are tested barefoot and without holding a handrail. Among various other parameters, stride width, step length, stance phase and foot rotation are measured. Both methods used reportedly have a high intra- and inter-observer reliability. The advantage of these highly accurate techniques is that they offer an objective and very detailed perspective on changes in the patient&#39;s posture and gait. Due to the amount of data generated, these techniques are, however, not so much suitable for everyday routine use, but rather interesting to scientifically evaluate long term alterations in posture and gait in patients like for example after lumbar fusion surgery.

Here, we present a protocol to analyze the posture and the gait of patients after lumbar fusion surgery by means of high-resolution video rasterstereography and a treadmill equipped with an integrated sensor mat. Allowing critical functional postoperative evaluation on a less subjective level may enhance accuracy and reliability of indication for surgery.

This protocol provides guidance on how to perform high resolution video rasterstereography and treadmill gait analysis on patients after lumbar fusion ...

Structured Motor Rehabilitation After Selective Nerve Transfers

After severe nerve injuries, selective nerve transfers provide an opportunity to restore motor and sensory function. Functional recovery depends both on the successful re-innervation of the targets in the periphery and on the motor re-learning process entailing cortical plasticity. While there is an increasing number of methods to improve rehabilitation, their routine implementation in a clinical setting remains a challenge due to their complexity and long duration. Therefore, recommendations for rehabilitation strategies are presented with the aim of guiding medical doctors and therapists through the long-lasting rehabilitation process and providing step-by-step instructions for supporting motor re-learning.
Directly after nerve transfer surgery, no motor function is present, and therapy should focus on promoting activity in the sensory-motor cortex areas of the paralyzed body part. After about two to six months (depending on the severity and modality of injury, the distance of nerve regeneration and many other factors), the first motor activity can be detected via electromyography (EMG). Within this phase of rehabilitation, multimodal feedback is used to re-learn the motor function. This is especially critical after nerve transfers, as muscle activation patterns change due to the altered neural connection. Finally, muscle strength should be sufficient to overcome gravity/resistance of antagonistic muscles and joint stiffness, and more functional tasks can be implemented in rehabilitation.

Here, we present a protocol for the motor rehabilitation of patients with severe nerve injuries and selective nerve transfer surgery. It aims at restoring the motor function proposing several stages in patient education, early-stage therapy after surgery and interventions for rehabilitation after successful re-innervation of the nerve&#8217;s target.

After severe nerve injuries, selective nerve transfers provide an opportunity to restore motor and sensory function. Functional recovery depends both on ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Combining Augmented Reality and 3D Printing to Display Patient Models on a Smartphone

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) printing (3DP) opens new possibilities in clinical applications. Although these technologies have grown exponentially in recent years, their adoption by physicians is still limited, since they require extensive knowledge of engineering and software development. Therefore, the purpose of this protocol is to describe a step-by-step methodology enabling inexperienced users to create a smartphone app, which combines AR and 3DP for the visualization of anatomical 3D models of patients with a 3D-printed reference marker. The protocol describes how to create 3D virtual models of a patient&rsquo;s anatomy derived from 3D medical images. It then explains how to perform positioning of the 3D models with respect to marker references. Also provided are instructions for how to 3D print the required tools and models. Finally, steps to deploy the app are provided. The protocol is based on free and multi-platform software and can be applied to any medical imaging modality or patient. An alternative approach is described to provide automatic registration between a 3D-printed model created from a patient&rsquo;s anatomy and the projected holograms. As an example, a clinical case of a patient suffering from distal leg sarcoma is provided to illustrate the methodology. It is expected that this protocol will accelerate the adoption of AR and 3DP technologies by medical professionals.

Presented here is a method to design an augmented reality smartphone application for the visualization of anatomical three-dimensional models of patients using a 3D-printed reference marker.

Augmented reality (AR) has great potential in education, training, and surgical guidance in the medical field. Its combination with three-dimensional (3D) ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

Fertility Sparing Procedure using Carbon Dioxide Fiber Laser Vaporization of Ovarian Endometrioma

The surgical management of endometrioma is still a matter of debate. Cystectomy, which is recognized as the standard technique, seems to be associated with a potential reduction in the ovarian reserve due to the inadvertent removal and thermal damage of healthy ovarian tissue. New ablative techniques with reduced tissue penetration depth and less thermal spread to the surrounding parenchyma may represent a viable alternative to cystectomy. For these reasons, the aim of this manuscript is to demonstrate the ablation of the endometrioma capsule using a CO2 fiber laser technique and discuss the clinical outcomes. Once the cyst has been drained and washed, a biopsy is taken. After cyst eversion, vaporization of the inner surface of the cyst is performed using a CO2 fiber laser. The technique is simple and reproducible as even young surgeons without any surgical experience were more confident in performing laser CO2 vaporization instead of cystectomy. The positive effects of CO2 technology are reported in a randomized controlled trial, where the postoperative changes in the antral follicular count (AFC) and antimullerian hormone (AMH) levels were compared between patients who had their endometrioma excised (cystectomy) and those who had undergone endometrioma vaporization with CO2 laser. The patients treated with CO2 laser showed significantly increased AFC without a reduction in serum AMH levels as compared to the cystectomy group, in which both parameters were significantly reduced. The postoperative pregnancy rate was also assessed, and comparable pregnancy rates were found after both treatments. On the contrary, patients treated with the CO2 fiber laser technique had more favorable in-vitro fertilization (IVF) outcomes compared to cystectomy.
In conclusion, the CO2 fiber laser technique may represent a viable alternative to cystectomy in the surgical treatment of endometrioma in terms of ovarian preservation, pregnancy rates, and IVF outcomes. Moreover, it has the advantage of being independent of the surgeon's skills and personal experience.

In this protocol, CO2 fiber laser technique is demonstrated for the surgical treatment of ovarian endometriosis, which represents a viable alternative in terms of fertility preservation with the major advantage of not being dependent on the surgeon's skills and personal experience.

The surgical management of endometrioma is still a matter of debate. Cystectomy, which is recognized as the standard technique, seems to be associated ...