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>

<ol><li>Committee on Adolescent Health Care. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ACOG+Committee+Opinion+No.+728%3A+M%C3%BCllerian+Agenesis%3A+Diagnosis%2C+Management%2C+And+Treatment%5BTitle%5D)+AND+%22Obstetrics+and+Gynecology%22%5BJournal%5D)">ACOG Committee Opinion No. 728: Müllerian Agenesis: Diagnosis, Management, And Treatment.</a> Obstetrics and Gynecology. 131 (1), 35-42 (2018).</li>
<li>Reichman, D. E., Laufer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mayer-Rokitansky-K%C3%BCster-Hauser+syndrome%3A+Fertility+counseling+and+treatment%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">Mayer-Rokitansky-Küster-Hauser syndrome: Fertility counseling and treatment.</a> Fertility and Sterility. 94 (5), 1941-1943 (2010).</li>
<li>Ben-Rafael, Z., Bar-Hava, I., Levy, T., Orvieto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simplifying+ovulation+induction+for+surrogacy+in+women+with+Mayer-Rokitansky-Kuster-Hauser+syndrome%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Simplifying ovulation induction for surrogacy in women with Mayer-Rokitansky-Kuster-Hauser syndrome.</a> Human Reproduction. 13 (6), 1470-1471 (1998).</li>
<li>Beski, S., Gorgy, A., Venkat, G., Craft, I. L., Edmonds, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gestational+surrogacy%3A+a+feasible+option+for+patients+with+Rokitansky+syndrome%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Gestational surrogacy: a feasible option for patients with Rokitansky syndrome.</a> Human Reproduction. 15 (11), 2326-2328 (2000).</li>
<li>Brännström, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Livebirth+after+uterus+transplantation%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Livebirth after uterus transplantation.</a> Lancet. 385 (9968), 607-616 (2015).</li>
<li>Sreenivas, K., Campo-Engelstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Domestic+and+international+surrogacy+laws%3A+implications+for+cancer+survivors%5BTitle%5D)+AND+%22Cancer+Treatment+and+Research%22%5BJournal%5D)">Domestic and international surrogacy laws: implications for cancer survivors.</a> Cancer Treatment and Research. 156, 135-152 (2010).</li>
<li>Soderstrom-Anttila, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surrogacy%3A+outcomes+for+surrogate+mothers%2C+children+and+the+resulting+families-a+systematic+review%5BTitle%5D)+AND+%22Human+Reproductive+Update%22%5BJournal%5D)">Surrogacy: outcomes for surrogate mothers, children and the resulting families-a systematic review.</a> Human Reproductive Update. 22 (2), 260-276 (2016).</li>
<li>Wood, E. G., Batzer, F. R., Corson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovarian+response+to+gonadotrophins%2C+optimal+method+for+oocyte+retrieval+and+pregnancy+outcome+in+patients+with+vaginal+agenesis%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Ovarian response to gonadotrophins, optimal method for oocyte retrieval and pregnancy outcome in patients with vaginal agenesis.</a> Human Reproduction. 14 (5), 1178-1181 (1999).</li>
<li>Raziel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surrogate+in+vitro+fertilization+outcome+in+typical+and+atypical+forms+of+Mayer-Rokitansky-Kuster-Hauser+syndrome%5BTitle%5D)+AND+%22Human+Reproduction%22%5BJournal%5D)">Surrogate in vitro fertilization outcome in typical and atypical forms of Mayer-Rokitansky-Kuster-Hauser syndrome.</a> Human Reproduction. 27 (1), 126-130 (2012).</li>
<li>Egarter, C., Huber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Successful+stimulation+and+retrieval+of+oocytes+in+patient+with+Mayer-Rokitansky-K%C3%BCster+syndrome%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Successful stimulation and retrieval of oocytes in patient with Mayer-Rokitansky-Küster syndrome.</a> Lancet. 1 (8597), 1283(1988).</li>
<li>Candiani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oocyte+retrieval+during+laparoscopic+vaginoplasty+to+reduce+invasiveness+in+the+treatment+of+Mayer-Rokitansky-K%C3%BCster-Hauser+syndrome%5BTitle%5D)+AND+%22Journal+of+Minimally+Invasive+Gynecology%22%5BJournal%5D)">Oocyte retrieval during laparoscopic vaginoplasty to reduce invasiveness in the treatment of Mayer-Rokitansky-Küster-Hauser syndrome.</a> Journal of Minimally Invasive Gynecology. 27 (1), 74-79 (2020).</li>
<li>Fedele, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creation+of+a+neovagina+by+Davydov%27s+laparoscopic+modified+technique+in+patients+with+Rokitansky+syndrome%5BTitle%5D)+AND+%22American+Journal+of+Obstetrics+and+Gynecology%22%5BJournal%5D)">Creation of a neovagina by Davydov's laparoscopic modified technique in patients with Rokitansky syndrome.</a> American Journal of Obstetrics and Gynecology. 202 (1), 1-6 (2010).</li>
<li>ESHRE Working Group on Ultrasound in ART et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recommendations+for+good+practice+in+ultrasound%3A+oocyte+pick+up%5BTitle%5D)+AND+%22Human+Reproduction+Open%22%5BJournal%5D)">Recommendations for good practice in ultrasound: oocyte pick up.</a> Human Reproduction Open. 2019 (4), (2019).</li>
<li>Callens, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+update+on+surgical+and+non-surgical+treatments+for+vaginal+hypoplasia%5BTitle%5D)+AND+%22Human+Reproductive+Update%22%5BJournal%5D)">An update on surgical and non-surgical treatments for vaginal hypoplasia.</a> Human Reproductive Update. 20 (5), 775-801 (2014).</li>
<li>Sönmezer, M., Gülümser, Ç, Sönmezer, M., Sükür, Y. E., Atabekoğlu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transabdominal+ultrasound+guided+oocyte+retrieval+using+vaginal+ultrasound+probe%3A+Definition+of+the+technique%5BTitle%5D)+AND+%22The+Journal+of+Obstetrics+and+Gynaecology+Research%22%5BJournal%5D)">Transabdominal ultrasound guided oocyte retrieval using vaginal ultrasound probe: Definition of the technique.</a> The Journal of Obstetrics and Gynaecology Research. 47 (2), 800-806 (2021).</li>
<li>Raju, G. A., Haranath, G. B., Krishna, K. M., Prakash, G. J., Madan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Successful+pregnancy+with+laparoscopic+oocyte+retrieval+and+in+vitro+fertilization+in+Mullerian+agenesis%5BTitle%5D)+AND+%22Singapore+Medicine+Journal%22%5BJournal%5D)">Successful pregnancy with laparoscopic oocyte retrieval and in vitro fertilization in Mullerian agenesis.</a> Singapore Medicine Journal. 47 (4), 329-331 (2006).</li>
<li>Liszewska-Kapłon, M., Strózik, M., Kotarski, Ł, Bagłaj, M., Hirnle, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mayer-Rokitansky-K%C3%BCster-Hauser+syndrome+as+an+interdisciplinary+problem%5BTitle%5D)+AND+%22Advances+in+Clinical+and+Experimental+Medicine%22%5BJournal%5D)">Mayer-Rokitansky-Küster-Hauser syndrome as an interdisciplinary problem.</a> Advances in Clinical and Experimental Medicine. 29 (4), 505-511 (2020).</li>
<li>Wagner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+management+during+the+adolescent+transition+period+of+girls+and+young+women+with+Mayer-Rokitansky-K%C3%BCster-Hauser+syndrome+%28MRKHS%29%3A+A+systematic+literature+review%5BTitle%5D)+AND+%22Orphanet+Journal+of+Rare+Diseases%22%5BJournal%5D)">Treatment management during the adolescent transition period of girls and young women with Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS): A systematic literature review.</a> Orphanet Journal of Rare Diseases. 11 (1), 152(2016).</li>
<li>Bean, E. J., Mazur, T., Robinson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mayer-Rokitansky-K%C3%BCster-Hauser+syndrome%3A+sexuality%2C+psychological+effects%2C+and+quality+of+life%5BTitle%5D)+AND+%22Journal+of+Pediatric+and+Adolescent+Gynecology%22%5BJournal%5D)">Mayer-Rokitansky-Küster-Hauser syndrome: sexuality, psychological effects, and quality of life.</a> Journal of Pediatric and Adolescent Gynecology. 22 (6), 339-346 (2009).</li>
</ol>

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 unfeasible due to extrapelvic ovaries located laterally along the pelvic walls14 or in proximity of organs such as the liver and gallbladder9, or due to the presence of pelvic kidneys. In these patients, a transabdominal approach could be hypothesized and has been reported in the literature: nonetheless, a mean number of 4.92 &#177; 1.7 oocytes was collected15, suggesting a suboptimal retrieval due to technical limitations. In addition, a transabdominal approach might be difficult or impossible in some cases, such as in patients with marked abdominal wall thickness or poor visibility of ovaries due to overlying loops of bowel16.
Safety is a noteworthy strength of this approach. In addition to the advantages of optimal visualization and access described above, combining two procedures into a single one reduces anesthesiologic risks. Of note, a similar operative time is maintained compared to that of Davydov laparoscopic vaginoplasty alone, for which a mean duration of 125 min was reported in the literature12. Moreover, the fact that patients undergo COS at a very young age rather than later in life does not imply an increased risk of ovarian hyperstimulation syndrome for them, as this complication is efficiently avoided by triggering final follicular maturation with a Gn-RH analog.
Current guidelines for MRKHS require clinicians to address future options for having children with patients and parents at the time of diagnosis as a means to help them cope with the disease and its implications1. Adoption, surrogacy, or uterine transplant are the available solutions, with both options for biological motherhood requiring oocyte retrieval17. This combined laparoscopic approach might thus also be particularly beneficial in those MRKHS patients expressing major distress about their diagnosis of infertility18. In fact, surveys have shown that infertility can be one of the most challenging conditions to accept for patients receiving the diagnosis of MRKHS19. Early cryopreservation of oocytes in patients with a desire for future genetic offspring might alleviate their psychological distress, compared to delaying this necessary invasive intervention to an undefined date in the future.
However, as the main limitation to this approach, not all patients who opt for oocyte cryopreservation as early as at the time of vaginoplasty will eventually pursue their desire for genetic motherhood in their later adulthood years. For this reason, the team should also obtain and record the patient&#39;s decision regarding the use of her oocytes in the event of her ceasing to renovate cryopreservation (i.e., elimination, donation for research purposes, donation, or sell for heterologous reproduction where legal).
As a possible modification of this technique, we foresee the use of laparoscopic ultrasound probes, which could assist the infertility expert in puncturing the follicles that are located far from the ovarian surface. Lastly, we envisage that building expertise in laparoscopic oocyte retrieval for fertility preservation at the time of other procedures might allow the availability of this option. As an example, patients undergoing other laparoscopic procedures such as myomectomy and showing an indication to fertility preservation might benefit from this approach.

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><tbody><tr><td>&lt;strong&gt;Oocyte retrieval procedure&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;u&gt;Equipment&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; O&lt;sub&gt;2&lt;/sub&gt; 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>&lt;u&gt;Consumables&lt;/u&gt;</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&amp;rsquo;s Advantage Fertilization medium SAGE</td><td>Origio</td><td>1020</td><td></td></tr><tr><td>Quinn&amp;rsquo;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>&lt;strong&gt;Oocyte denudation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;u&gt;Equipment&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; O&lt;sub&gt;2&lt;/sub&gt; 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>&lt;u&gt;Consumables&lt;/u&gt;</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&amp;rsquo;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 &amp;mu;m)</td><td>Cook</td><td>K-FPIP-1140-10BS-5</td><td></td></tr><tr><td>Stripping pipette tips (170 &amp;mu;m)</td><td>Cook</td><td>K-FPIP-1170-10BS-5</td><td></td></tr><tr><td>&lt;strong&gt;Oocyte vitrification&lt;/strong&gt;</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 &amp;micro;L</td><td>Thermo Scientific</td><td>2160G</td><td></td></tr><tr><td>Tips 2-20 &amp;micro;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 oocyte survival rate following this vitrification protocol-defined as the proportion of morphologically intact oocytes at the time of intracytoplasmic sperm injection-was 84.5 &#177; 19.3. The total average operative time was 114 &#177; 17 min, intraoperative blood loss was insignificant (&#60;50 mL) in all patients, and no intraoperative adverse events were observed. The urinary catheter and vaginal gauze were removed on day 2 after surgery in all patients. On day 3 post surgery, the patients began daily dilator use and were discharged on day 6.0 &#177; 1.0. One postoperative complication occurred in one patient (fever developing on day 5 post surgery and intraperitoneal fluid detection on transabdominal ultrasound). The patient was treated successfully with orally administered antibiotics and discharged on day 9 post surgery. Follow-up after surgery confirmed anatomical and functional success of the procedure. None of the patients reported any limitation to their daily life activities, any pain, or urinary symptom.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63634/63634fig01.jpg" /> 
Figure 1: Oocyte vitrification protocol. Schematic showing the various steps of vitrification workflow of oocytes. Oocytes are first placed in a drop of WS1 (A), and then in a drop of WS2 (B). Mix the drop of ES1 into WS2 and after 2 min of incubation, merge the drop of ES2 to the previously merged drops (B). After 2 min, a third drop of ES3 is merged (B). After 1 min, place oocytes in a drop of ES4 incubating for 10 min (C). Then, oocytes are moved sequentially into the three drops of VS for 60 s (D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63634/63634fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Characteristic</td>
			<td>Mean &#177; SD or n (%)</td>
		</tr>
		<tr>
			<td colspan="2">Age at diagnosis, years</td>
			<td>13.8 &#177; 1.5</td>
		</tr>
		<tr>
			<td colspan="2">46 XX karyotype</td>
			<td>23</td>
		</tr>
		<tr>
			<td colspan="2">Age at surgery, years</td>
			<td>20.3 &#177; 3.4</td>
		</tr>
		<tr>
			<td colspan="2">Antral Follicle Count (AFC), n</td>
			<td>13.2 &#177; 4.1</td>
		</tr>
		<tr>
			<td colspan="2">Relevant findings (instrumental/laboratory)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Horseshoe kidney</td>
			<td>2 (8.7)</td>
		</tr>
		<tr>
			<td></td>
			<td>Extra pelvic ovary/ovaries</td>
			<td>3 (13.0)</td>
		</tr>
		<tr>
			<td></td>
			<td>Unilateral renal agenesis</td>
			<td>2 (8.7)</td>
		</tr>
		<tr>
			<td></td>
			<td>Dilative cardiomyopathy</td>
			<td>1 (4.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>Mesocardia</td>
			<td>1 (4.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>Vater Syndrome</td>
			<td>1 (4.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>Sensorineural hearing loss</td>
			<td>1 (4.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>Congenital clubfoot</td>
			<td>1 (4.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>Scoliosis</td>
			<td>1 (4.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>Celiac disease</td>
			<td>1 (4.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>Type 1 Diabetes</td>
			<td>1 (4.3)</td>
		</tr>
		<tr>
			<td></td>
			<td>Autoimmune Hypothyroidism</td>
			<td>1 (4.3)</td>
		</tr>
	</tbody>
</table>
Table 1: Demographic, anamnestic, and clinical data of the 23 patients treated so far.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Mean &#177; SD</td>
		</tr>
		<tr>
			<td>FSH starting dose, IU</td>
			<td>196 &#177; 44</td>
		</tr>
		<tr>
			<td>FSH total dose, IU</td>
			<td>2,174 &#177; 506</td>
		</tr>
		<tr>
			<td>Days of stimulation, n</td>
			<td>12.1 &#177; 0.4</td>
		</tr>
		<tr>
			<td>E2 levels at triggering, pg/mL</td>
			<td>4,330 &#177; 2,007</td>
		</tr>
		<tr>
			<td>P levels at triggering, ng/mL</td>
			<td>1.06&#160; &#177; 0.95</td>
		</tr>
	</tbody>
</table>
Table 2: Controlled ovarian stimulation data of the 23 patients treated so far. Abbreviations: FSH = follicle-stimulating hormone; E2 = estradiol; P = progesterone.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Mean &#177; SD or n (%)</td>
		</tr>
		<tr>
			<td>Operative time, min</td>
			<td>114 &#177; 17</td>
		</tr>
		<tr>
			<td>Hospital stay, days</td>
			<td>6.0 &#177; 1.0</td>
		</tr>
		<tr>
			<td>Blood loss, mL</td>
			<td>insignificant</td>
		</tr>
		<tr>
			<td>Retrieved oocytes, n</td>
			<td>11.4 &#177; 5.4</td>
		</tr>
		<tr>
			<td>Mature oocytes (vitrified), n</td>
			<td>9.6 &#177; 4.3</td>
		</tr>
		<tr>
			<td>Complications</td>
			<td></td>
		</tr>
		<tr>
			<td>None</td>
			<td>22 (95.6)</td>
		</tr>
		<tr>
			<td>Postoperative fever and intraperitoneal fluid detection</td>
			<td>1 (4.3)</td>
		</tr>
	</tbody>
</table>
Table 3: Outcomes of the combined laparoscopic procedure of oocyte retrieval and vaginoplasty in the 23 patients treated so far.

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

Nanyang Technological University

Research

JoVE Journal

Medicine

3.5K Views.  IRCCS San Raffaele Scientific Institute. Onze aanpak maakt het mogelijk om invasieve ziekte bij de behandeling van het Mayer-Rokitansky-Kuster-Hauser-syndroom te minimaliseren door laparoscopische vaginoplastiek en het ophalen van eicellen te combineren voor toekomstige vruchtbaarheidsbehandelingen. De belangrijkste voordelen van onze aanpak zijn de vereiste voor een enkele anesthesie en het overwinnen van de onhaalbaarheid van een transvaginale oöcyt-retrieval bij jongere Rokitansky-patiënten. De procedure zal worden gedemonstree...

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 ...

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) is still the only option to preserve fertility in women who need to start gonadotoxic treatment urgently or in prepubertal children. During ovarian cortex preparation for cryopreservation, medullar tissue is removed. Growing antral follicles reside at the border of the cortex-medullar interface of the ovary and are broken during this process, releasing their cumulus-oocyte complex (COC). By thoroughly inspecting the medium and fragmented medullar tissue, these immature cumulus-oocyte complexes can be identified without interfering with the OTC procedure. The ovarian tissue-derived immature oocytes can be successfully matured in vitro, creating an additional source of gametes for fertility preservation. If OTC is performed within or near a medical assisted reproduction laboratory, all necessary in vitro maturation (IVM) and oocyte vitrification tools can be at hand. Furthermore, upon remission and child wish, the patient has multiple options for fertility restoration: ovarian tissue transplantation or embryo transfer after the insemination of vitrified/warmed oocytes. Hence, ovarian tissue oocyte-in vitro maturation (OTO-IVM) can be a valuable adjunct fertility preservation technique.

Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation

Presented here is ovarian tissue oocyte-in vitro maturation (OTO-IVM), an accessible technique within a medical assisted reproduction (MAR) laboratory offering realistic additional fertility preservation options to patients who need ovarian tissue cryopreservation.

Mature oocyte vitrification is the standard of care to preserve fertility in women at risk of infertility. However, ovarian tissue cryopreservation (OTC) ...

Developmental Biology

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

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 ...

Cryopreservation of Oocytes Retrieved from Ovarian Tissue to Optimize Fertility Preservation in Prepubertal Girls and Women

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of premature ovarian insufficiency (POI) in the context of urgent gonadotoxic treatments or ovarian surgery. Fertility preservation is challenging because there is no consensus regarding patient management, preservation fertility strategies, or even technical laboratory protocols, which implies that each procedure must be adapted to the characteristics of the patient profile and its own risk-benefit ratio. During OTC, mature/immature oocytes can be aspirated directly from large/small antral follicles within ovarian tissue samples and/or be released into culture media from growing follicles during ovarian tissue dissection in prepubertal girls and women. In this manuscript, we present a protocol that combines ovarian tissue freezing with the cryopreservation of mature/immature oocytes retrieved from ovarian tissue samples, improving the reproductive potential of fertility preservation. Appropriate collection, handling, and storage of ovarian tissue and oocytes before, during, and after the cryopreservation will be described. The subsequent use and safety of cryopreserved/thawed ovarian tissue samples and oocytes will also be discussed, as well as the optimal timing for in vitro maturation of immature oocytes. We recommend the systematic use of this protocol in fertility preservation of prepubertal girls and women as it increases the whole reproductive potential of fertility preservation (i.e., oocyte vitrification in addition of OTC) and also improves the safety and use of fertility preservation (i.e., thawing of oocytes versus ovarian graft), maximizing the chance of successful childbirth for the patients at risk of POI.

We propose a protocol for fertility preservation in prepubertal girls and women at risk of premature ovarian insufficiency. It combines ovarian tissue freezing and cryopreservation of oocytes retrieved from ovarian tissue. This strategy improves the safety and optimizes the reproductive potential of fertility preservation, maximizing the chance of childbirth.

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of ...

Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation is recognized by several international scientific societies as the gold standard for fertility preservation in postpubertal women, for both medical and non-medical indications. The main medical indications are oncologic diseases, gynecologic diseases such as severe endometriosis, systemic diseases compromising the ovarian reserve, and genetic conditions involving premature menopause. This paper describes the whole clinical and laboratory work-up of a fertility preservation treatment by outlining recommendations for objective and evidence-based counseling. Furthermore, it focuses on the effectiveness of the procedure and describes the most appropriate strategies to fully exploit the ovarian reserve and maximize the number of oocytes retrieved in the shortest possible time. The evaluation of the ovarian reserve, the definition of an ideal stimulation protocol, as well as oocyte retrieval, denudation, and vitrification procedures have been detailed along with approaches to maximize their efficacy, efficiency, and safety.

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

Preserving female fertility is crucial in a multifunctional healthcare system that takes care of patients' future quality of life. Oocyte cryopreservation ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

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

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

Follicular Viability Assessment in Vitrified Human Ovarian Cortex Tissues

Vitrification of Ovarian Cortex Tissue to Achieve a Glassy State of Aggregation

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or for pre-pubertal girls, it is often the only option for fertility protection. Cryopreservation can be performed either by vitrification or by slow freezing. Slow freezing is currently the standard approach. An increasing number of studies indicate that vitrification can replace slow freezing in the state-of-the-art in vitro fertilization (IVF) laboratories, significantly improving thawing survival rates and simplifying the technical aspects of cryopreservation. A metal grid-based, high-throughput protocol for rapid vitrification of ovarian cortex tissue, suitable for clinical routine, is described. The sterilization of metal grids and liquid nitrogen ensures high quality, meeting good manufacturing practice (GMP) standards. Vitrification was conducted to ensure ultra-rapid cooling rates. Instead of slowly thawing, samples were rapidly warmed. To assess follicular viability, calcein staining was performed both prior to cryopreservation and after rapid warming. The successful application of vitrification and rapid warming using metal grids is reported. No significant differences in follicular viability were observed prior to vitrification and after rapid warming. These results substantiate the high capacity of tissue vitrification for clinical routine applications as a potential substitute for the widely used slow-freezing method.

Author Spotlight: Advancing Fertility Preservation in Young Female Cancer Patients Through Ovarian Tissue Vitrification and In Vitro Follicular Growth

A protocol for the vitrification of ovarian tissue, as an alternative cryopreservation method to the widely used slow freezing protocol, is presented.

Ovarian tissue cryopreservation (OTC) is an important option for fertility preservation. For patients whose gonadotoxic treatments cannot be postponed or ...

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 ...

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining&#160;natural immunodeficiency up to day 17 postfertilization, making it ideal to study&#160;short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation

Here, we describe a protocol for developing a chick chorioallantoic membrane (CAM) xenografting model for human ovarian tissue and demonstrate the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle ...

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

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