No specific funding was received for this work.

The local ethical committee at the tertiary referral center for MRKHS (IRCCS San Raffaele University Hospital, Milan, Italy) was notified and approved the protocol before its implementation in July 2017. All patients or guardians gave signed informed consent for laparoscopic oocyte retrieval and cryopreservation during vaginoplasty and for the use of anonymized clinical/laboratory data for scientific purposes.
1. Team composition
<ol>
	<li>Designate a dedicated team, composed of an experienced laparoscopic surgeon, an experienced vaginal surgeon, an infertility expert, a dedicated psychologist, and a committed nurse.</li>
	<li>Assist the patient as a team during the whole setup and performance of the procedure.</li>
</ol>
2. Diagnostic work-up and counseling
<ol>
	<li>Perform transabdominal pelvic and abdominal ultrasonography on the patient for visualization of the ovaries and estimation of the Antral Follicle Count (AFC). Assess blood levels of anti-M&#252;llerian-hormone (AMH) to optimize the gonadotrophin starting dose for controlled ovarian stimulation. Perform serological tests for infectious diseases (HIV-Ab, HCV-Ab, HBV-Ab, RPR-TPHA) according to national/local protocols for cryopreservation of tissues/gametes.</li>
	<li>Counsel the patient about both the sexual and reproductive aspects of MRKHS, including nonsurgical and surgical vaginoplasty, the costs and legislation related to surrogacy, the perspective of uterine transplantation in the context of an experimental program, the different approaches available for oocyte retrieval and the performance of oocyte cryopreservation in terms of live-birth rates.</li>
	<li>Offer the patient psychological evaluation to support her through the procedure and assess emotional stability and commitment and desire for future motherhood. Obtain signed, informed consent for concomitant laparoscopic oocyte retrieval, gamete cryopreservation, and laparoscopic vaginoplasty. In the case of an underage patient, perform the clinical assessments in the presence of the parents who also sign the relative informed consent.</li>
</ol>
3. Scheduling of the surgical procedure and of controlled ovarian stimulation
<ol>
	<li>Schedule the procedure of concomitant laparoscopic oocyte retrieval, gamete cryopreservation, and laparoscopic vaginoplasty, taking into account the availability of the clinical team (see above) as well as that of the lab staff performing oocyte vitrification.</li>
	<li>Start controlled ovarian stimulation (COS) on day -14 from the day of surgery, taking into account a planned duration of COS of n = 12 days and the 36 h needed between ovulation triggering and oocyte retrieval and vitrification.</li>
	<li>In case COS requires a different duration from the expected, reschedule the surgery accordingly.</li>
</ol>
4. Controlled ovarian stimulation: starting dose, dose adjustments and monitoring, final oocyte maturation triggering
<ol>
	<li>Start COS in any phase of the ovarian cycle, as commonly done in the context of &#34;random start&#34; protocols for fertility preservation.</li>
	<li>Choose the starting dose of follicle-stimulating hormone (FSH)/human menopausal gonadotropin (hMG) based on the patient&#39;s AFC and AMH, and instruct the patient to continue daily self-administered subcutaneous injections. Perform individualized dose adjustments subsequently based on the ovarian response.</li>
	<li>Monitor ovarian response by serial transabdominal ultrasound and concomitant measurements of E2 and P (day 1, day 6, day 8, day 10, and day 12).</li>
	<li>Trigger final oocyte maturation by subcutaneous administration of 0.2 mL of a Gn-RH-analog (Triptorelin).</li>
</ol>
5. Clinical procedure (laparoscopy): oocyte retrieval
<ol>
	<li>Position the patient in modified dorsal lithotomy position, which allows excellent simultaneous access to the abdomen and perineum. Induce general anesthesia and administer 2 g of Cefazolin intravenously as per routine intraoperative antibiotic prophylaxis. Position an intravesical Foley catheter.</li>
	<li>Establish pneumoperitoneum with a peri-umbilical Veress needle, position one 10 mm umbilical trocar for insertion of the laparoscope and two 5 mm trocars in the right and left upper quadrants. Under laparoscopic vision, use a 17 G single lumen needle connected to an aspiration pump, as commonly employed for transvaginal retrieval, through the right and left trocars for puncture of the right and left ovary, respectively.</li>
	<li>Lift and hold the ovaries with laparoscopic forceps to facilitate exposure of the follicles. When aspirating multiple follicles that are one close to another, retain the needle tip in the ovary to reduce the number of times that the ovarian cortex is transfixed and the inherent risk of bleeding.</li>
</ol>
6. Clinical procedure (laparoscopy): vaginoplasty
NOTE: Vaginoplasty steps remain unchanged compared to the Davydov&#39;s laparoscopic modified technique12.
<ol>
	<li>Dissect the peritoneum by lifting and incising the peritoneal strand transversally between the two rudimental uterine horns, and then extending the incision anteriorly, laterally, and posteriorly.</li>
	<li>Begin a purse-string suture in polydioxanone synthetic absorbable (PDS) 2-0 monofilament in each hemipelvis, from the mobilized peritoneum above the bladder, with consecutive transfixion of the round ligament, the tubal isthmus, the uteroovarian ligament, and the lateral peritoneal leaf. Then, include in the two sutures the lateral aspect of the mesorectum and the anterior aspect of the rectal serosa, immediately below the rectosigmoid junction.</li>
	<li>Expose the vaginal dimple on the perineal approach and perform an H-shaped incision, and create a neovaginal space by sharp and blunt dissection of the vesicorectal space. Then, pull down the dissected peritoneal margins to the edge of the vaginal vestibulum. Position interrupted sutures in PDS 3-0 for the peritoneal-vestibular anastomosis. Lastly, place a paraffin coated gauze in the peritoneum-coated neovagina.</li>
</ol>
7. IVF laboratory: the day before the oocyte retrieval
<ol>
	<li>Prepare 2 to 5 round-bottom tubes with 9 mL of Quinn&#39;s Advantage Medium with HEPES (supplemented with 5% human serum albumin [HSA]) according to the number of expected follicles and incubate them overnight at 37 &#176;C.</li>
	<li>Prepare one or two 4-well dishes with 0.5 mL/well of Fertilization Medium (supplemented with 5% HSA), covered with 0.5 mL of mineral oil for embryo culture and incubate it overnight at 37 &#176;C in a controlled atmosphere (6% CO2, 5% O2).</li>
	<li>Prepare one dish containing 9 (30 &#181;L) drops of Continuous Single Culture (CSCM) medium (supplemented with 10% serum substitute supplement [SSS]) covered with 6 mL of mineral oil. Incubate it overnight at 37 &#176;C in a controlled atmosphere (6% CO2, 5% O2).</li>
</ol>
8. IVF laboratory: oocyte retrieval procedure
<ol>
	<li>Prepare a solution of Quinn&#39;s Advantage Medium with HEPES (supplemented with 5% HSA) with heparin at a final concentration of 10 IU/mL in which follicular aspirates will be collected.</li>
	<li>Similar to routine transvaginal oocyte retrieval13, collect the follicular aspirates in 10 mL round-bottom tubes, kept warm (37 &#176;C) in a portable incubator and immediately transported to the embryology laboratory, with a transportation time of approximately 10 min.</li>
	<li>Examine the follicular fluid in a prewarmed sterile 90 mm Petri dish to identify cumulus-oocyte complexes (COC). Once a COC is detected, rinse it in a central-well dish filled with 1 mL of prewarmed Quinn&#39;s Advantage Medium with HEPES (supplemented with 5% HSA).</li>
	<li>As all the COCs are collected, place them into the 4-well dish containing the Fertilization Medium and label the lid and the bottom with data relating to the patient&#39;s identity.</li>
	<li>Incubate them at 37 &#176;C in a controlled atmosphere (6% CO2, 5% O2).</li>
	<li>Ensure that a double-checking of the procedure is performed by a second member of the laboratory staff.</li>
</ol>
9. Oocyte denudation
<ol>
	<li>Perform cumulus/corona cells removal within 38 h post ovulation trigger.</li>
	<li>Prepare a solution of Quinn&#39;s Advantage Medium with HEPES (supplemented with 5% HSA) with Hyaluronidase at a final concentration of 10 IU/mL.</li>
	<li>Prepare a central-well dish with 1 mL of prewarmed Quinn&#39;s Advantage Medium with HEPES (supplemented with 5% HSA) containing Hyaluronidase and another one with 1 mL of prewarmed HEPES-buffered medium (supplemented with 5% HSA).</li>
	<li>Label the oocyte denudation dishes with patient identification data.</li>
	<li>Place COCs in the central-well containing the enzyme to disperse the cumulus cells with a glass Pasteur pipette, gently pipetting the solution containing COCs up and down for up to 30 s.</li>
	<li>Move the oocytes to the second central-well dish containing only HEPES-buffered medium, making sure to displace a minimum amount of the enzyme.</li>
	<li>Remove the remaining corona cells using denuding pipettes with decreasing inner diameters (170-140 &#181;m).</li>
	<li>Assess the stage of oocyte maturation, separating metaphase II (MII) oocytes, characterized by the extrusion of the first polar body, from metaphase I oocytes (MI) and germinal vesicles (GV).</li>
	<li>Transfer the MII oocytes in an IVF culture 60 mm Petri dish containing nine (30 &#181;L) drops of Continuous Single Culture (CSCM) medium (supplemented with 10% serum substitute supplement [SSS]) covered with 6 mL of mineral oil.</li>
	<li>Incubate them at 37 &#176;C in a controlled atmosphere (6% CO2, 5% O2).</li>
</ol>
10. Oocyte vitrification
<ol>
	<li>Perform vitrification of MII oocytes immediately after oocyte denudation, following the protocol provided by the vitrification kit&#39;s manufacturer.</li>
	<li>Bring to room temperature (25-27 &#176;C): the equilibration solution (ES), the vitrification solution (VS), and the washing solution (WS), nearly 30 min before the procedure. 
	NOTE: As reported by the manufacturers, ES contains 7.5% DMSO, 7.5% ethylene glycol, 20% dextran serum supplement (DSS), Gentamicin in M-199 HEPES-buffered Medium; VS contains 15% DMSO, 15% ethylene glycol, 0.5 M sucrose, 20% DSS, Gentamicin in an M-199 HEPES-buffered Medium, and WS contains 20% DSS, Gentamicin in an M-199 HEPES-buffered Medium.</li>
	<li>For a cryodevice, use Cryotop consisting of a fine strip of transparent film attached to a plastic handle.</li>
	<li>Label the cryodevices with the patient&#39;s name, date of birth, ID, date of cryopreservation, and number of oocytes loaded on any single device.</li>
	<li>Label the vitrification dish with the patient&#39;s name and ID.</li>
	<li>Ensure that a witness operator checks the correct patient data on the cryodevices and dish.</li>
	<li>Fill a cooling box up to the top with fresh liquid nitrogen and cover until use to minimize dispersion and evaporation.</li>
	<li>Use a stripper pipette with an inner diameter of 170 &#181;m to avoid damaging oocytes during manipulation.</li>
	<li>Gently shake each vial of ES, VS, and WS to mix the contents before use.</li>
	<li>Prepare the lid of a 60 mm Petri dish with one drop of 50 &#181;L of WS1 (Figure 1A).</li>
	<li>Place drops just before use to limit medium evaporation.</li>
	<li>Take the oocyte dish from the incubator and transfer the MII oocytes (up to 3 at a time) with minimal volume of medium from the culture dish into the 50 &#181;L of WS.</li>
	<li>Dispense 50 &#181;L drops of WS2, ES1, and ES2 in close proximity and transfer oocytes from drop WS1 into drop WS2 (Figure 1B).</li>
	<li>Merge the drop of ES1 to WS2 and wait for 2 min for the spontaneous mixing of both the solutions.</li>
	<li>Then, merge the drop of ES2 to the previously merged drops and leave for further 2 min.</li>
	<li>Finally, merge a new 100 &#181;L drop of ES3 to the previously merged drops and leave for 1 additional minute.</li>
	<li>Place the oocytes into a 100 &#181;L drop of ES4 for 10 min (Figure 1C).</li>
	<li>Dispense two 50 &#181;L drops of VS and one 100 &#181;L of VS (Figure 1D).</li>
	<li>Move the oocytes sequentially into the three drops of VS for 60 s so that the oocytes remain in each drop for ~20 s.</li>
	<li>When approximately 10 s are left before the end of the 60 s of incubation, place the cryodevice under the microscope.</li>
	<li>Carry the oocytes at the tip of the pipette and place them on the cryodevice with the minimum amount of VS.</li>
	<li>Aspirate the excess of VS, leaving the oocytes covered by a thin layer of VS.</li>
	<li>Plunge the cryodevice directly into liquid nitrogen and move it rapidly. Keep the device in liquid nitrogen and cover it with the protective lid.</li>
	<li>Store the cryodevice in a storage system (e.g., visiotube) and place it in the cryogenic tank. Record cryopreservation data on the laboratory database and sheet.</li>
</ol>
11. Inpatient postoperative care
<ol>
	<li>Remove the urinary catheter and vaginal paraffin-coated gauze 48 h after surgery.</li>
	<li>Digitally explore the patients&#39; neovagina after the removal and instruct the patient on how to perform the digital exploration. Then, coat a vaginal mold (9 cm in length and 2 cm in diameter) with estrogen gel (to favor epithelialization) and instruct the patient on how to insert and maintain the mold.</li>
	<li>Starting on day 3 post surgery and on each following day, instruct the patient on how to position the mold and maintain it in place for at least 2 h daily.</li>
	<li>If no complications occur, discharge the patient on day 5-6 post surgery.</li>
</ol>
12. Outpatient postoperative care
<ol>
	<li>Instruct the patient on how to perform neovaginal dilation for 2 months with the same mold (9 cm in length and 2 cm in diameter) used during the hospital stay, and then a larger one (11 cm in length and 2.5 cm in diameter).</li>
	<li>After the 3 months&#39; visit, allow the patient to begin sexual activity and inform her to keep using the molds on the days when there is no intercourse.</li>
</ol>
13. Follow-up
<ol>
	<li>Schedule follow-up visits at 1, 3, 6, and 12 months post procedure.</li>
	<li>At each follow-up visit, assess compliance with dilation exercises and ask the patient whether she has experienced any limitation to her daily life activities, pain, urinary symptoms, or sexual dysfunction (as of the visit after the third month post surgery). Perform gynecological examination at each follow-up visit to measure vaginal width, length, suspension, and mobility of the adnexa.</li>
</ol>

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The authors do not have any conflicts of interest to disclose.

This protocol reduces the invasiveness in the treatment of MRKHS by combining the procedures of vaginoplasty and oocyte retrieval. To this purpose, it is crucial that a dedicated team is designated to ensure that the timing of COS, the surgical procedure, and oocyte vitrification are scheduled efficiently.
MRKHS patients for whom this combined laparoscopic method is expected to be most beneficial are those in whom a transvaginal retrieval would be considered technically challenging or unfeasib...

With an incidence of approximately 1 in 4-10,000 women, MRKHS is the cause of 15% of primary amenorrhea cases. MRKHS is characterized by the congenital absence of the upper segment of the vagina and the uterus, whereas urinary tract and skeletal anomalies are associated variably. More specifically, a vaginal vault with a depth of 1-2 cm is usually present, and two rudimental uterine horns may be found1.
In the past, the primary medical interest in MRKHS was to enable normal sexual intercourse, which generally requires the construction of a neovagina by either non-surgical or surgical approaches2. However, advances in reproductive medicine currently allow genetic motherhood in MRKHS patients, by either surrogacy3,4 or-more recently-uterus transplantation5. While uterus transplantation is still an experimental procedure, surrogacy is available in many countries worldwide6, and reported obstetric and psychosocial outcomes are comparable to those of standard in vitro fertilization and oocyte donation7.
Both surrogacy and uterus transplantation require oocyte retrieval and in vitro fertilization, but no consensus exists on how to perform egg collection in patients with MRKHS. A transvaginal approach can be unfeasible because of insufficient vaginal elasticity8 even after vaginoplasty, atypical location of the ovaries9, or excessive distance between the ovaries and the vaginal cuff4,10. In these cases, laparoscopic oocyte retrieval represents the optimal approach in terms of surgical access. However, as most MRKHS patients currently undergo laparoscopic neovaginoplasty, we suggest performing a laparoscopic oocyte retrieval at the time of surgery for vaginoplasty11, followed by oocyte cryopreservation for future use, thus minimizing invasiveness while combining the treatment of the sexual and reproductive function of MRKHS patients.
Demographic data of patients 
Twenty-three MRKH patients underwent treatment with this protocol so far. Anamnestic, instrumental, and laboratory findings of patients are summarized in Table 1. Unless specified in Table 1, no associated congenital anomalies were found.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Oocyte retrieval procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 O2 Incubator</td>
			<td>Sanyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td>Cooper Surgical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Portable incubator</td>
			<td>Cooper Surgical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL Polystyrene Round-Bottom Tube</td>
			<td>Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well dish</td>
			<td>Nunc</td>
			<td>144444</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Nunc</td>
			<td>FA9150270</td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm Petri dish</td>
			<td>Nunc</td>
			<td>FA9150360</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin 100 mg/ml in Normal Saline (5%)</td>
			<td>Origio</td>
			<td>3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil for embryo culture</td>
			<td>Origio</td>
			<td>4008</td>
			<td></td>
		</tr>
		<tr>
			<td>One Well Dish</td>
			<td>Oosafe</td>
			<td>OOPW-CW05</td>
			<td></td>
		</tr>
		<tr>
			<td>Quinn&#8217;s Advantage Fertilization medium SAGE</td>
			<td>Origio</td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Quinn&#8217;s Advantage medium with HEPES</td>
			<td>Origio</td>
			<td>1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile glass pasteur pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte denudation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 O2 Incubator</td>
			<td>Sanyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flexipet adjustable handle set</td>
			<td>Cook</td>
			<td>G18674</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td>Cooper Surgical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-well dish</td>
			<td>Nunc</td>
			<td>144444</td>
			<td></td>
		</tr>
		<tr>
			<td>CSCM (Continuos single culture) medium</td>
			<td>Fujifilm irvine Scientific</td>
			<td>90165</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Albumin 100 mg/mL in Normal Saline (5%)</td>
			<td>Origio</td>
			<td>3001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Fujifilm Irvine Scientific</td>
			<td>90101</td>
			<td></td>
		</tr>
		<tr>
			<td>IVF culture 60 mm petri dish</td>
			<td>Nunc</td>
			<td>FA9150270</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil for embryo culture</td>
			<td>Origio</td>
			<td>4008</td>
			<td></td>
		</tr>
		<tr>
			<td>One Well Dish</td>
			<td>Oosafe</td>
			<td>OOPW-CW05</td>
			<td></td>
		</tr>
		<tr>
			<td>Quinn&#8217;s Advantage medium with HEPES</td>
			<td>Origio</td>
			<td>1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum Substitute Supplement</td>
			<td>Fujifilm irvine Scientific</td>
			<td>99193</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile glass pasteur pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stripping pipette tips (140 &#956;m)</td>
			<td>Cook</td>
			<td>K-FPIP-1140-10BS-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Stripping pipette tips (170 &#956;m)</td>
			<td>Cook</td>
			<td>K-FPIP-1170-10BS-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte vitrification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dish</td>
			<td>NUNC</td>
			<td>150255</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>NUNC</td>
			<td>150270</td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm Petri dish</td>
			<td>NUNC</td>
			<td>150360</td>
			<td></td>
		</tr>
		<tr>
			<td>Container for Cooling rack</td>
			<td>Kitazato</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryodevice/cryotop</td>
			<td>Kitazato</td>
			<td>81111</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic timer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flexipet</td>
			<td>COOK</td>
			<td>K- 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson Pipetman</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Printer LabXpert</td>
			<td>Brady</td>
			<td>XSL-86-461</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 20-200 &#181;L</td>
			<td>Thermo Scientific</td>
			<td>2160G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 2-20 &#181;L</td>
			<td>Thermo Scientific</td>
			<td>2139-HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Visotubes</td>
			<td>Cryo Bio System</td>
			<td>20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrification Freeze Kit</td>
			<td>Fujifilm Irvine Scientific</td>
			<td>90133-SO</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitrification Thaw kit</td>
			<td>Fujifilm Irvine Scientific</td>
			<td>90137-SO</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Table 2 includes ovarian stimulation data of the patients, whereas main surgical and functional outcomes are described in Table 3. The concomitant laparoscopic procedures of oocyte retrieval and vaginoplasty were combined successfully in all patients. An average of 11.4 &#177; 5.4 (mean &#177; SD) oocytes were retrieved, and 9.6 &#177; 4.3 MII oocytes were cryopreserved (Table 3). In our experience with oocyte cryopreservation in patients undergoing ART, postwarming oocy...

Watch this Scientific Journal Video about Laparoscopic Oocyte Retrieval and Cryopreservation during Vaginoplasty for Treatment of Mayer-Rokitansky-Kuster-Hauser Syndrome at JoVE.com

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

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, ...

laparoscopic-oocyte-retrieval-cryopreservation-during-vaginoplasty

Research

JoVE Journal

Medicine

3.4K Views.  IRCCS San Raffaele Scientific Institute. 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.

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

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Transaxillary First Rib Resection for Treatment of the Thoracic Outlet Syndrome

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol has been used for the decompression of trapped neurovascular structures in the TOS. Among the other surgical procedures, the advantage of the TFRR is that it has the smallest rate of recurrence and better cosmetic outcomes. The disadvantage of TFRR is that it provides a narrow, and deep working corridor that makes obtaining vascular control challenging.

Here, we present a protocol of the transaxillary resection of the first rib for treatment of thoracic outlet syndrome caused by compression of the brachial plexus, subclavian vein and artery.

Thoracic outlet syndrome (TOS) is a common disorder that causes a significant loss of productivity. The transaxillary first rib resection (TFRR) protocol ...

Technical Modification of the Terminal Ureter During Total Transperitoneal Laparoscopic Nephroureterectomy for Upper Urinary Tract Urothelial Carcinoma

Upper tract urothelial carcinoma (UTUC) accounts for 5%&ndash;10% of all urothelial tumors. Radical nephroureterectomy is the standard treatment procedure. At present, different choices still exist for treating the ureteral end during laparoscopic ureteral bladder sleeve resection. Our center has adopted a new method for treating the ureteral end. This new method can increase the operating space and reduce the difficulty of the surgery compared with current methods.

Here, we present a protocol to increase the surgical field of view and reduce the difficulty of total transperitoneal laparoscopic nephroureterectomy surgery by precutting the umbilical ligament before treating the terminal ureter.

Upper tract urothelial carcinoma (UTUC) accounts for 5%&ndash;10% of all urothelial tumors. Radical nephroureterectomy is the standard treatment ...

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.

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 ...

Surgical Techniques to Optimize Ovarian Reserve during Laparoscopic Cystectomy for Ovarian Endometrioma

Surgical management of ovarian endometrioma in patients desiring fertility is complicated by the need to balance maximal resection of disease with efforts to spare normal ovarian cortex. Optimization of tubal anatomy is another frequent consideration. Fertility-sparing laparoscopic techniques at the time of cystectomy for ovarian endometrioma seek to limit iatrogenic surgical damage to the ovarian cortex and strategically assess and respond to genital tract patency. Surgical candidates frequently desire relief from endometriosis-associated pain while also seeking to optimize spontaneous or assisted conception rates. Operative benefits include potential for surgical and histopathologic diagnosis of endometriosis, evaluation of genital tract patency, and treatment of visualized lesions. Resection of ovarian endometrioma nonetheless poses significant risks, including surgical injury, blood loss, post-surgical decline in ovarian reserve and post-operative inflammation with adhesion formation, both of which may impair folliculogenesis.
We present the case of a 32-year-old woman with known endometriosis and continued pain refractory to medical management who opted for surgical management of her disease tailored toward optimizing her chances at future conception. Using this case as an example, we describe techniques and considerations for diagnostic laparoscopy, adhesiolysis, ovarian cystectomy, chromopertubation, and salpingectomy with a focus on maintaining a fertility-preserving approach.

This protocol presents techniques to laparoscopically excise ovarian endometrioma, to perform adhesiolysis with sparing electrosurgical application, and to employ intraoperative chromopertubation to assess for genital tract patency. This systematic approach will facilitate optimal endometriosis management, guide concomitant adnexal surgeries, and enhance post-surgical fertility outcomes.

Surgical management of ovarian endometrioma in patients desiring fertility is complicated by the need to balance maximal resection of disease with efforts ...

Laparoscopic Non-Mesh Cerclage Pectopexy for Pelvic Organ Prolapse

Pelvic organ prolapse (POP) is widespread among the female population and significantly impairs the patient's quality of life. It is important to restore apical support for treating POP. Sacrocolpopexy and pectopexy are indicated for apical prolapse. Using a synthetic mesh in these techniques increases success by enhancing apical support. However, the implantation of synthetic mesh is associated with mesh-related complications. In addition, the exorbitant cost of synthetic mesh and lack of universal access limit the popularity of these procedures. The current study develops a unique technique known as laparoscopic non-mesh cerclage pectopexy (LNMCP), in which permanent cervical cerclage sutures are embedded in the round ligament until the iliopectineal ligament. The iliopectineal ligament was sutured, resulting in a firm cervical suspension. The procedure was successfully performed in 16 cases in the hospital. The surgical duration was 67.8 min &plusmn; 15.5 min, and the blood loss was 73.1 mL &plusmn; 51.1 mL. No procedural complications were seen. LNMCP is associated with an objective success rate of 100% and a subjective success rate of 93.8%. LNMCP for patients with apical prolapse obviates the need for a mesh, thereby avoiding complications associated with mesh erosion and reducing medical costs. In addition, it is easy to perform even in resource-poor areas without access to synthetic mesh.

The present pilot study describes the development of laparoscopic non-mesh cerclage pectopexy to treat pelvic organ prolapse. The procedure can be used to prevent any complications associated with the use of mesh.

Pelvic organ prolapse (POP) is widespread among the female population and significantly impairs the patient's quality of life. It is important to restore ...

Advancing Cryostorage of Human Stem Cell-Derived Retinal Pigment Epithelial Cells

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of&#160;RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2&#8242;-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Author Spotlight: Development of an Efficient and Feasible Method of Retinal Pigment Epithelial Cell Freezing

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...

Laparoscopic Retroperitoneal Treatment of Necrotizing Pancreatitis Complications

Retroperitoneal Laparoscopic Debridement and Drainage for Pancreatic Abscess

In patients with severe necrotizing pancreatitis, pancreatic necrosis and secondary infection of surrounding tissues can quickly spread to the whole retroperitoneal space. Treatment of pancreatic abscess complicating necrotizing pancreatitis is difficult and has a high mortality rate. The well-accepted treatment strategy is early debridement of necrotic tissues, drainage, and postoperative continuous retroperitoneal lavage. However, traditional open surgery has several disadvantages, such as severe trauma, interference with abdominal organs, a high rate of postoperative infection and adhesion, and hardness with repeated debridement. The retroperitoneal laparoscopic approach has the advantages of minimal invasion, a better drainage route, convenient repeated debridement, and avoidance of the spread of retroperitoneal infection to the abdominal cavity. In addition, retroperitoneal drainage leads to fewer drainage tube problems, including miscounting, displacement, or siphon. The debridement and drainage of pancreatic abscess tissue via the retroperitoneal laparoscopic approach plays an increasingly irreplaceable role in improving patient prognosis and saving healthcare resources and costs. The main procedures described here include laying the patient on the right side, raising the lumbar bridge and then arranging the trocar; establishing the pneumoperitoneum and cleaning the pararenal fat tissues; opening the lateral pyramidal fascia and the perirenal fascia outside the peritoneal reflections; opening the anterior renal fascia and entering the anterior pararenal space from the rear; clearing the necrotic tissue and accumulating fluid; and placing drainage tubes and performing postoperative continuous retroperitoneal lavage.

Author Spotlight: Advancements in Retroperitoneal Approach for Necrotizing Pancreatitis

To improve the drainage of patients with necrotizing pancreatitis complicated with peripancreatic abscess and reduce the mortality of patients with necrotizing pancreatitis, we adopted a retroperitoneal approach for peripancreatic abscess debridement and drainage by laparoscopy.

In patients with severe necrotizing pancreatitis, pancreatic necrosis and secondary infection of surrounding tissues can quickly spread to the whole ...

An Efficient Modified Method for Treating Wasting Marmoset Syndrome

Modification of the Treatment Methods for Wasting Marmoset Syndrome with Tranexamic Acid and Supportive Measures

Wasting marmoset syndrome (WMS), a serious disease in captive common marmoset (Callithrix jacchus) colonies, is associated with a high mortality rate. The specific cause of WMS is still unclear and there are few effective treatments. Previously, we had reported a tranexamic acid therapy with supportive measures as a useful treatment for WMS. In the present study, we describe the modified method: a combination of 0.1 mL of 5% tranexamic acid subcutaneously five times per week, 2.0 mL of amino acid formulation intravenously three times per week, 5.0 mL of Ringer's lactate with 0.1 mL of a vitamin formulation subcutaneously three times per week, and oral administration of 0.1 mL of an iron formulation five times per week. We also describe how to administer the solution intravenously via the saphenous vein with a tip of restraining the animal, as well as the detailed methods for oral and subcutaneous administration. The modified methods have comparable efficiency to the original WMS treatment method.

Author Spotlight: Developing a Safer and More Efficient Treatment Protocol for Wasting Marmoset Syndrome (WMS)

Here, we describe the modified treatment methods for wasting marmoset syndrome (WMS, also known as inflammatory bowel disease (IBD)-like disease) with tranexamic acid. We also present how to administer the therapeutic agents orally, subcutaneously, and intravenously.

Wasting marmoset syndrome (WMS), a serious disease in captive common marmoset (Callithrix jacchus) colonies, is associated with a high mortality rate. The ...

Improving IVF Outcomes with Partial Granulosa Cell Removal Post-Retrieval

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

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients continue to experience fertilization disorders, leading to low fertilization rates and the production of nonviable embryos. Short-term fertilization involves the early removal of granulosa cells to observe the extrusion of the second polar body, enabling the assessment of fertilization and early remedial measures to address low fertilization rates and complete fertilization failure. However, the observation of short-term fertilization in IVF is impeded by challenges such as excessively large unprocessed clusters of cumulus-oocyte complexes and adhesion between eggs, necessitating complex external procedures for granulosa cell removal, and prolonged observation duration. To tackle this issue, this study proposes a method of immediate partial removal of granulosa cells post egg retrieval. This approach streamlines subsequent stages of short-term fertilization and traditional fertilization monitoring, reduces the time needed for oocyte extrusion, minimizes the likelihood of external environmental impacts on the oocytes, and decreases the risk of missing the critical fertilization observation window. Consequently, the study yields novel clinical evidence aimed at enhancing the operational efficiency of embryonic laboratories in IVF.

Author Spotlight: Evaluating the Impact of Immediate Partial Removal of Cumulus-Oocyte Complexes on Fertilization Efficiency and Embryo Quality

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.

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients ...