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

Summary

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.

Abstract

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 °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.

Introduction

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 surrogacy^3,4 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 cuff^4,10. 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.

Protocol

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

1. Designate a dedicated team, composed of an experienced laparoscopic surgeon, an experienced vaginal surgeon, an infertility expert, a dedicated psychologist, and a committed nurse.
2. Assist the patient as a team during the whole setup and performance of the procedure.

2. Diagnostic work-up and counseling

1. 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-Müllerian-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.
2. 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.
3. 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.

3. Scheduling of the surgical procedure and of controlled ovarian stimulation

1. 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.
2. 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.
3. In case COS requires a different duration from the expected, reschedule the surgery accordingly.

4. Controlled ovarian stimulation: starting dose, dose adjustments and monitoring, final oocyte maturation triggering

1. Start COS in any phase of the ovarian cycle, as commonly done in the context of "random start" protocols for fertility preservation.
2. Choose the starting dose of follicle-stimulating hormone (FSH)/human menopausal gonadotropin (hMG) based on the patient'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.
3. 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).
4. Trigger final oocyte maturation by subcutaneous administration of 0.2 mL of a Gn-RH-analog (Triptorelin).

5. Clinical procedure (laparoscopy): oocyte retrieval

1. 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.
2. 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.
3. 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.

6. Clinical procedure (laparoscopy): vaginoplasty

NOTE: Vaginoplasty steps remain unchanged compared to the Davydov's laparoscopic modified technique¹².

1. 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.
2. 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.
3. 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.

7. IVF laboratory: the day before the oocyte retrieval

1. Prepare 2 to 5 round-bottom tubes with 9 mL of Quinn'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 °C.
2. 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 °C in a controlled atmosphere (6% CO₂, 5% O₂).
3. Prepare one dish containing 9 (30 µL) 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 °C in a controlled atmosphere (6% CO₂, 5% O₂).

8. IVF laboratory: oocyte retrieval procedure

1. Prepare a solution of Quinn'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.
2. Similar to routine transvaginal oocyte retrieval¹³, collect the follicular aspirates in 10 mL round-bottom tubes, kept warm (37 °C) in a portable incubator and immediately transported to the embryology laboratory, with a transportation time of approximately 10 min.
3. 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's Advantage Medium with HEPES (supplemented with 5% HSA).
4. 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's identity.
5. Incubate them at 37 °C in a controlled atmosphere (6% CO₂, 5% O₂).
6. Ensure that a double-checking of the procedure is performed by a second member of the laboratory staff.

9. Oocyte denudation

1. Perform cumulus/corona cells removal within 38 h post ovulation trigger.
2. Prepare a solution of Quinn's Advantage Medium with HEPES (supplemented with 5% HSA) with Hyaluronidase at a final concentration of 10 IU/mL.
3. Prepare a central-well dish with 1 mL of prewarmed Quinn'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).
4. Label the oocyte denudation dishes with patient identification data.
5. 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.
6. Move the oocytes to the second central-well dish containing only HEPES-buffered medium, making sure to displace a minimum amount of the enzyme.
7. Remove the remaining corona cells using denuding pipettes with decreasing inner diameters (170-140 µm).
8. 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).
9. Transfer the MII oocytes in an IVF culture 60 mm Petri dish containing nine (30 µL) drops of Continuous Single Culture (CSCM) medium (supplemented with 10% serum substitute supplement [SSS]) covered with 6 mL of mineral oil.
10. Incubate them at 37 °C in a controlled atmosphere (6% CO₂, 5% O₂).

10. Oocyte vitrification

1. Perform vitrification of MII oocytes immediately after oocyte denudation, following the protocol provided by the vitrification kit's manufacturer.
2. Bring to room temperature (25-27 °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.
3. For a cryodevice, use Cryotop consisting of a fine strip of transparent film attached to a plastic handle.
4. Label the cryodevices with the patient's name, date of birth, ID, date of cryopreservation, and number of oocytes loaded on any single device.
5. Label the vitrification dish with the patient's name and ID.
6. Ensure that a witness operator checks the correct patient data on the cryodevices and dish.
7. Fill a cooling box up to the top with fresh liquid nitrogen and cover until use to minimize dispersion and evaporation.
8. Use a stripper pipette with an inner diameter of 170 µm to avoid damaging oocytes during manipulation.
9. Gently shake each vial of ES, VS, and WS to mix the contents before use.
10. Prepare the lid of a 60 mm Petri dish with one drop of 50 µL of WS1 (Figure 1A).
11. Place drops just before use to limit medium evaporation.
12. 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 µL of WS.
13. Dispense 50 µL drops of WS2, ES1, and ES2 in close proximity and transfer oocytes from drop WS1 into drop WS2 (Figure 1B).
14. Merge the drop of ES1 to WS2 and wait for 2 min for the spontaneous mixing of both the solutions.
15. Then, merge the drop of ES2 to the previously merged drops and leave for further 2 min.
16. Finally, merge a new 100 µL drop of ES3 to the previously merged drops and leave for 1 additional minute.
17. Place the oocytes into a 100 µL drop of ES4 for 10 min (Figure 1C).
18. Dispense two 50 µL drops of VS and one 100 µL of VS (Figure 1D).
19. Move the oocytes sequentially into the three drops of VS for 60 s so that the oocytes remain in each drop for ~20 s.
20. When approximately 10 s are left before the end of the 60 s of incubation, place the cryodevice under the microscope.
21. Carry the oocytes at the tip of the pipette and place them on the cryodevice with the minimum amount of VS.
22. Aspirate the excess of VS, leaving the oocytes covered by a thin layer of VS.
23. Plunge the cryodevice directly into liquid nitrogen and move it rapidly. Keep the device in liquid nitrogen and cover it with the protective lid.
24. 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.

11. Inpatient postoperative care

1. Remove the urinary catheter and vaginal paraffin-coated gauze 48 h after surgery.
2. Digitally explore the patients' 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.
3. 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.
4. If no complications occur, discharge the patient on day 5-6 post surgery.

12. Outpatient postoperative care

1. 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).
2. After the 3 months' visit, allow the patient to begin sexual activity and inform her to keep using the molds on the days when there is no intercourse.

13. Follow-up

1. Schedule follow-up visits at 1, 3, 6, and 12 months post procedure.
2. 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.

Results

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 ± 5.4 (mean ± SD) oocytes were retrieved, and 9.6 ± 4.3 MII oocytes were cryopreserved (Table 3). In our experience with oocyte cryopreservation in patients undergoing ART, postwarming oocy...

Discussion

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...

Materials

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