Ovarian Tissue Oocyte-In Vitro Maturation for Fertility Preservation
Summary
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.
Abstract
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.
Introduction
Fertility preservation (FP) options for women planned for gonadotoxic treatment, sex-reassignment therapy, or women who have a genetic predisposition for premature ovarian failure, depend on the health and age of the patient, available timeframe, type of treatment, patient's preference, and FP procedures available at the fertility center of choice. Vitrification of mature oocytes obtained after ovarian stimulation with gonadotropins and oocyte retrieval in a medical assisted reproduction (MAR) laboratory cycle is considered the preferred option for FP1,2. However, for prepubertal girls, women in whom the urgent start of gonadotoxic treatment or gonadectomy is required, or women with a high risk of permanent amenorrhea due to gonadotoxic treatment, a cycle of ovarian stimulation with gonadotropins is not possible, and ovarian tissue cryopreservation (OTC), which is an accepted and valid technique for FP1,2,3, is the only option. The goal of OTC is to cryopreserve thousands of dormant primordial follicles in the ovarian cortex tissue, which can resume growth after the transplantation of frozen/thawed tissue onto the remaining ovary or in a peritoneal pocket after the careful screening of minimal residual disease in representative tissue fragments.
In order to obtain cortical fragments of 1-2 mm thickness suitable for cryopreservation, the soft medullar tissue needs to be removed. This medullar tissue typically entails growing follicles in various stages of development that escape the stiff ovarian cortex to allow for their growth and expansion4. For many years, several labs have been investigating the potential of these oocytes recovered from follicles residing in the remnant medullar tissue after ovarian cortical fragment preparation using in vitro maturation (IVM)5,6,7, referred to as ovarian tissue oocyte IVM (OTO-IVM). Antral follicles, even those less than 6 mm in diameter, contain immature oocytes surrounded by cumulus cells that can mature, fertilize, and develop into healthy babies using an IVM system8,9. IVM is considered the standard of care for women at risk for ovarian hyperstimulation syndrome (OHSS), such as polycystic ovary syndrome (PCOS) patients. However, in the field of FP, there are limited data available for IVM in cases with a contraindication for ovarian stimulation; IVM of oocytes collected transvaginally is still considered innovative, and OTO-IVM is considered experimental2,10. That said, the reports of the first live births after OTO-IVM11,12,13 highlight the potential of using OTO-IVM as an add-on technique when OTC is required for FP in patients14.
This study provides technical details to adopt OTO-IVM in the MAR laboratory and illustrates the results obtained in a single center.
Protocol
Representative Results
Discussion
The priority of the FP procedure is always to manipulate and freeze the ovarian cortex according to the standard operating protocol that has been validated in the clinic. A drawback in FP is the absence of a standard protocol available in the published literature regarding OTC and OTO-IVM. It is difficult to assess the efficiency and validity of the techniques and adaptations since there is a large time gap between freezing/vitrification and thawing/warming in a clinical setting. If changes to the OTC protocol are made t…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was conducted at the IVF laboratory of Brussels IVF, Universitair Ziekenhuis of VUB, Brussels. The authors would like to thank all Brussels IVF laboratory team members for their high skills, accuracy, and flexibility needed to establish a fertility preservation unit within a MAR laboratory.
Materials
| 1000 µL filter tips | Eppendorf/VWR International | 613-6780 | COC search |
| Benchtop Cooler | Fisher Scientific | 15-350-54 | Benchtop Cooler lid is used to prepare the tissue, Benchtop Cooler tube holder to keep cryovials with freezing medium cooled |
| Corning Cell culture dish, non-treated, 100 mm | Corning/VWR International | 430591 | Dish for ovarian tissue preparation |
| CryoSure-DMSO | WAK Chemicals | 0482 | Cryoprotectant for ovarian tissue cryopreservation |
| Cumulase | Origio/CooperSurgical | 16125000A | recombinant human hyaluronidase enzyme for cumulus cell removal after IVM |
| Decontamination spray: Suprox | Medipure LTD | MP016 | Desinfectant solution for aseptic handling with bactericide and sporicide action |
| Disposable scalpels | Swann-Morton | 0511 | Ovarian tissue preparation |
| Falcon 14 mL Round Bottom Polystyrene Test Tube, with Snap Cap, Sterile | Falcon/VWR International | BDAA352057 | Medium container |
| Falcon Cell strainer 70 µm | Falcon/VWR International | 352350 | Filter for elimination of red blood cell contamination and COC search |
| Freeze control Ampoule Cryochamber and Temperature Controller | Cryologic | CL-8800i CC60AS | Slow freezing machine |
| FSH: Menopur 75 IU | Ferring | BE197504 | Follicle Stimulating Hormone : Supplement for IVM medium |
| Handling pipette 290-310 µm | Vitrolife | 15538 | COC search: gentle transfer of COC without damaging oocyte-cumulus cell connectivity |
| hCG: Brevactid 5000 IE | Ferring | 5008001036 | Human Chorionic Gonadotropin : Supplement for IVM medium |
| High security tube | CryoBioSystem | 022252 | cryovial, heat-sealed for safe cryostorage |
| HSA-solution | Vitrolife | 10064 | Human serum Albumin: supplement for IVM medium |
| Leibovitz's L-15 medium | Life Technologies Europe | 31415-029 | Handling medium for ovarian tissue preparation |
| MediCult IVM system | Origio/CooperSurgical | 82214010 | medium for IVM containing both LAG and IVM medium. IVM medium needs to be supplemented as detailed in the protocol |
| METZENBAUM fino scissors 140 mm | Chirurgical Maintenance | VIZ08280314 | Medium size scissors for initial medulla removal |
| Nunc 4-well dishes for IVF | Nunc/VWR International | 144444 | COC collection during COC search and IVM culture |
| Nunc Invitro fertilization Petri Dish with Vented Lid, 60 mm, Non-Pyrogenic, Sterile | Thermo Scientific/VWR | NUNC150270 | Dish for COC search |
| Oocyte handling medium : Flushing Medium with heparin | Origio/CooperSurgical | 10765060 | Search medium for COC search |
| Ovoil | Vitrolife | 10029 | oil for IVM culture |
| Penicillin/Streptomicin mix | Life Technologies Europe | 15140-148 | Supplement for OTC handling medium |
| Scissors, curved, 150 mm long, 20 mm blade | Chirurgical Maintenance | VIREBST999-SC | Small size scissors for residual medulla removal |
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