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 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 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, 20...

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 ...

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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 ...

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. ...

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 ...

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 ...

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

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, we propose that a concomitant laparoscopic oocyte retrieval for cryopreservation is performed. Oocyte retrieval is pursued at the beginning of the laparoscopy. Right and left 5 mm trocars are positioned, through which a 17 G ovum aspiration needle is used for puncture of the right and left ovaries, respectively. To facilitate exposure of the follicles, the ovaries are mobilized and held with laparoscopic forceps.
When aspirating multiple follicles near each other, the needle tip is retained in the ovary to reduce the number of times that the ovarian cortex is transfixed and due to the inherent risk of bleeding. Subsequent steps are unchanged compared to the Davydov laparoscopic modified technique for vaginoplasty. Prior to surgery, controlled ovarian stimulation is performed with a gonadotropin hormone-releasing hormone (Gn-RH) antagonist protocol, and the concomitant procedure of oocyte retrieval and vaginoplasty is scheduled 36 h after the final follicular maturation trigger. Follicular fluid is collected in the same 10 mL sterile tubes used during transvaginal oocyte retrieval and transferred in a warming block (37 &#176;C) to the assisted reproduction laboratory, where mature (metaphase II) oocytes are vitrified.
In this case, a series of 23 women with MRKH, oocytes were successfully retrieved and cryopreserved in all patients; vaginoplasty was subsequently conducted without modifications, and the inpatient and outpatient postoperative care (day of urinary catheter removal, day of hospital discharge, dilator use, and comfort at follow-up) remained unaffected. One postoperative complication occurred in one patient (fever developing on day 5 post surgery and intraperitoneal fluid detection on transabdominal ultrasound) and resolved after conservative treatment. Rather than performing surgical vaginoplasty and delaying oocyte retrieval in MRKH patients, this approach combines both procedures in a single laparoscopy, thereby minimizing surgical invasiveness and anesthesiologic risks.

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.

In Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) patients who are scheduled for laparoscopic vaginoplasty and have a desire for biological motherhood, ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Acupoint Needle-Embedding Combined with Ironing Therapy for Postoperative Pain After Anal Surgery

Acupoint needle-embedding combined with ironing therapy is a non-drug treatment method to release postoperative pain after anal surgery. The practice is guided by traditional Chinese medicine (TCM) syndrome differentiation theory and employs acupoint stimulation and heat to alleviate pain. Although prior research has shown that these are dependable methods for pain relief, the combined effect of the two techniques has not been described. In our research, we found that compared to using diclofenac sodium enteric-coated capsules alone, adding acupoint needle-embedding combined with ironing therapy was more effective for reducing pain levels at different stages after hemorrhoid surgery. Although this technique is efficient and commonly used in clinics, due to its invasive practice, acupoint needle embedding still carries risks relating to hospital-acquired infections and broken needles. Ironing therapy, on the other hand, can result in burns and connective tissue injuries. Therefore, there is an urgent need to develop a standardized protocol for medical staff. Our protocol refines the traditional techniques and provides detailed instructions on patient preparation, operation techniques, and postoperative care to ensure the therapy is carried out safely and efficiently. By standardizing this therapy, this technique is expected to become an important complementary therapy for postoperative pain relief&#160;in hemorrhoids, which will significantly improve patients &#39;life quality after anal surgery.

The high incidence of pain after anal surgery is due to the physiological structure and pathological status of the anus. At present, the use of painkillers cannot provide satisfactory pain relief, which makes complementary therapy important. This paper proposes acupoint needle-embedding combined with ironing therapy to relieve postoperative pain.

Acupoint needle-embedding combined with ironing therapy is a non-drug treatment method to release postoperative pain after anal surgery. The practice is ...