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, compose...

<ol>
	<li>Committee on Adolescent Health Care. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACOG+Committee+Opinion+No.+728:+M&#252;llerian+Agenesis:+Diagnosis,+Management,+And+Treatment.">ACOG Committee Opinion No. 728: M&#252;llerian 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mayer-Rokitansky-K&#252;ster-Hauser+syndrome:+Fertility+counseling+and+treatment.">Mayer-Rokitansky-K&#252;ster-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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplifying+ovulation+induction+for+surrogacy+in+women+with+Mayer-Rokitansky-Kuster-Hauser+syndrome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gestational+surrogacy:+a+feasible+option+for+patients+with+Rokitansky+syndrome.">Gestational surrogacy: a feasible option for patients with Rokitansky syndrome.</a> Human Reproduction. 15 (11), 2326-2328 (2000).</li><li>Br&#228;nnstr&#246;m, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Livebirth+after+uterus+transplantation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domestic+and+international+surrogacy+laws:+implications+for+cancer+survivors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surrogacy:+outcomes+for+surrogate+mothers,+children+and+the+resulting+families-a+systematic+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+response+to+gonadotrophins,+optimal+method+for+oocyte+retrieval+and+pregnancy+outcome+in+patients+with+vaginal+agenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surrogate+in+vitro+fertilization+outcome+in+typical+and+atypical+forms+of+Mayer-Rokitansky-Kuster-Hauser+syndrome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+stimulation+and+retrieval+of+oocytes+in+patient+with+Mayer-Rokitansky-K&#252;ster+syndrome.">Successful stimulation and retrieval of oocytes in patient with Mayer-Rokitansky-K&#252;ster syndrome.</a> Lancet. 1 (8597), 1283 (1988).</li><li>Candiani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte+retrieval+during+laparoscopic+vaginoplasty+to+reduce+invasiveness+in+the+treatment+of+Mayer-Rokitansky-K&#252;ster-Hauser+syndrome.">Oocyte retrieval during laparoscopic vaginoplasty to reduce invasiveness in the treatment of Mayer-Rokitansky-K&#252;ster-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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+a+neovagina+by+Davydov's+laparoscopic+modified+technique+in+patients+with+Rokitansky+syndrome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+good+practice+in+ultrasound:+oocyte+pick+up.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+update+on+surgical+and+non-surgical+treatments+for+vaginal+hypoplasia.">An update on surgical and non-surgical treatments for vaginal hypoplasia.</a> Human Reproductive Update. 20 (5), 775-801 (2014).</li><li>S&#246;nmezer, M., G&#252;l&#252;mser, &. #. 1. 9. 9. ;., S&#246;nmezer, M., S&#252;k&#252;r, Y. E., Atabeko&#287;lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transabdominal+ultrasound+guided+oocyte+retrieval+using+vaginal+ultrasound+probe:+Definition+of+the+technique.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+pregnancy+with+laparoscopic+oocyte+retrieval+and+in+vitro+fertilization+in+Mullerian+agenesis.">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-Kap&#322;on, M., Str&#243;zik, M., Kotarski, &. #. 3. 2. 1. ;., Bag&#322;aj, M., Hirnle, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mayer-Rokitansky-K&#252;ster-Hauser+syndrome+as+an+interdisciplinary+problem.">Mayer-Rokitansky-K&#252;ster-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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+management+during+the+adolescent+transition+period+of+girls+and+young+women+with+Mayer-Rokitansky-K&#252;ster-Hauser+syndrome+(MRKHS):+A+systematic+literature+review.">Treatment management during the adolescent transition period of girls and young women with Mayer-Rokitansky-K&#252;ster-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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mayer-Rokitansky-K&#252;ster-Hauser+syndrome:+sexuality,+psychological+effects,+and+quality+of+life.">Mayer-Rokitansky-K&#252;ster-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 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, ...

Our approach allows to minimize invasive sickness in the treatment of Mayer-Rokitansky-Kuster-Hauser syndrome by combining laparoscopic vaginoplasty and oocyte retrieval for future fertility treatments. The main advantages of our approach are the requirement for a single anesthesia and the overcoming of the infeasibility of a transvaginal oocyte retrieval in younger Rokitansky patients. The procedure will be demonstrated by Diana Delprato, a senior gynecologist of our infertility unit and by Alessandra Alteri and Greta Cermisoni, two senior clinical embryologists from our assisted reproduction lab.Start controlled ovarian stimulation on day 14 from the day of surgery. Monitor ovarian response by serial transabdominal ultrasound and concomitant measurements of estradiol and progesterone. Establish pneumoperitoneum with an open technique.Position one 10-millimeter umbilical trocar for insertion of the laparoscope and three five-millimeter trocars in the central, right, and left lower quadrants. Under laparoscopic vision, use a 17-gauge single lumen needle connected to an aspiration pump, as commonly employed for transvaginal retrieval through the central and left trocars for puncture of the right and left ovary, respectively. 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. Dissect the peritoneum by lifting and incising the peritoneal strand transversely between the two rudimental uterine horns, and then, extending the incision laterally, anteriorly, and posteriorly. Expose the vaginal dimple on the perineal approach and perform an H-shaped incision and create a neo-vaginal 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 polydioxanone synthetic absorbable 3-0 for the peritoneal vestibular anastomosis. Begin a purse string suture in polydioxanone synthetic absorbable 2-0 monofilament in each hemipelvis from the mobilized peritoneum above the bladder with connective transfixion of the round ligament, the tubal isthmus, the utero-ovarian 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. Lastly, place a paraffin-coated gauze in the peritoneum-coated neovagina. Collect the follicular aspirates in 10-millimeter round-bottom tubes kept warm in a portable incubator and immediately transport them to the embryology laboratory with a transportation time of approximately 10 minutes.As all the cumulus oocyte complexes are collected, place them into the four-well dish containing the fertilization medium and label the lid and the bottom with data relating to the patient's identity. Incubate them at 37 degrees Celsius in a controlled atmosphere containing 6%carbon dioxide and 5%oxygen. Ensure that a double-checking of the procedure is performed by a second member of the laboratory staff.Perform cumulus corona cells removal within 38 hours post-ovulation trigger. Place cumulus oocyte complexes in the central well containing the enzyme to disperse the cumulus cells with a glass Pasteur pipette, gently pipetting the solution containing cumulus oocyte complexes up and down for up to 30 seconds. Move the oocytes to the second central well dish containing only HEPES buffered medium, making sure to displace a minimum amount of the enzyme.Remove the remaining corona cells using denuding pipettes with decreasing inner diameters. Assess the stage of oocyte maturation, separating metaphase II oocytes. Transfer the metaphase II oocytes in an IVF culture 60-millimeter Petri dish containing nine drops of continuous single culture medium covered with six millimeter of mineral oil.Incubate them at 37 degrees Celsius in a controlled atmosphere containing 6%carbon dioxide and 5%oxygen. Fill a cooling box up to the top with fresh liquid nitrogen and cover until used to minimize dispersion and evaporation. Use a stripper pipette with an inner diameter of 170 micrometers to avoid damaging oocytes during manipulation.Gently shake each vial of equilibration solution, vitrification solution, and washing solution to mix the contents before use. Prepare the lid of a 60-milliliter Petri dish with one drop of 50 microliters of washing solution one. Place drops just before use to limit medium evaporation.Take the oocyte dish from the incubator and transfer the metaphase II oocytes with a minimal volume of medium from the culture dish into the 50 microliters of washing solution. Dispense 50-microliter drops of washing solution two, equilibration solution one, and equilibration solution two in close proximity and transfer oocytes from drop washing solution one into drop washing solution two. Merge the drop of equilibration solution one to washing solution two and wait for two minutes for the spontaneous mixing of both the solutions.Then, merge the drop of equilibration solution two to the previously merged drops and leave for a further two minutes. Finally, merge a new 100-microliter drop of equilibration solution three to the previously merged drops and leave for one additional minute. Place the oocytes into 100-microliter drop of equilibration solution four for 10 minutes.Dispense two 50-microliter drops of vitrification solution and one 100-microliters of vitrification solution. Move the oocytes sequentially into the three drops of vitrification solution for 60 seconds so that the oocytes remain in each drop for approximately 20 seconds. When approximately 10 seconds are left before the end of the 60 seconds of incubation, place the cryodevice under the microscope.Carry the oocytes at the tip of the pipette and place them on the cryodevice with the minimum amount of vitrification solution. Aspirate the excess vitrification solution, leaving the oocytes covered by a thin layer of vitrification solution. Plunge the cryodevice directly into liquid nitrogen and move it rapidly.Keep the device in liquid nitrogen and cover it with a protective lid. The concomitant laparoscopic procedures of oocyte retrieval and vaginoplasty were combined successfully in all patients. An average of 11.4 5.4 oocytes were retrieved and 9.6 4.3 M2 oocytes were cryo-preserved.The total average operative time was 114 17 minutes. Intraoperative blood loss was insignificant in all patients and no intraoperative adverse events were observed. On day three post-surgery, the patients began daily dilator use and were discharged on day 6.0 1.0.A cautious aspiration of follicles within the ovary is recommended, with due attention paid to the automatic mobilization of the ovaries in order to ensure optimal access and retrieval.

laparoscopic-oocyte-retrieval-cryopreservation-during-vaginoplasty

Research

JoVE Journal

Medicine

3.3K visualizações.  IRCCS San Raffaele Scientific Institute. Nossa abordagem permite minimizar a doença invasiva no tratamento da síndrome de Mayer-Rokitansky-Kuster-Hauser, combinando vaginoplastia laparoscópica e recuperação de ovócitos para futuros tratamentos de fertilidade. As principais vantagens de nossa abordagem são a necessidade de uma única anestesia e a superação da inviabilidade de uma recuperação de ovócitos transvaginais em pacientes mais jovens de Rokitansky. O procedimento será demonstrado por Diana Delprato, ginecologist...