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Method Article
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 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.
The field of fertility preservation has grown over the last two decades due to the increasing number of patients at risk of premature ovarian insufficiency (POI)1,2,3. The current available medical options to preserve fertility are ovarian tissue cryopreservation (OTC)4, oocyte/embryo freezing after ovarian stimulation5, administration of GnRH analogues6, or ovarian transposition7. OTC is a major advance for fertility preservation, particularly in prepubertal girls, where it is the only option currently available to preserve fertility and also in women who cannot delay the onset of their gonadotoxic treatment2,4.
OTC allows the preservation of a high number of primordial follicles, which are located in the outer 1 mm of the ovarian cortex2. Frozen/thawed ovarian tissue can be subsequently used by graft (orthotopic or heterotopic, autologous or donor) or cultured in vitro to obtain mature oocytes2. The graft of frozen-thawed prepubertal ovarian tissue samples has been shown to induce puberty8,9. In women, the reproductive outcomes after orthotopic autografting of frozen-thawed ovarian cortex are reassuring, with live birth rates reaching 57.5% after natural conceptions10 and between 30% to 70% after Assisted Reproductive Techniques (ART) conceptions11. Since the first live birth from orthotopic transplantation of frozen/thawed human ovarian tissue in 200412, this technique allowed the birth of at least 130 children worldwide2. The hormonal and reproductive functions of the graft tissue generally last for several years11,13,14, confirming its long-term functionality.
However, auto-transplantation of ovarian tissue samples carries a theoretical risk of reintroducing viable malignant cells in some patients15,16,17,18, particularly in leukemia survivors19. To date, no case of transmission of cancer via the graft of frozen/thawed ovarian cortex has been reported in healthy cancer survivors11, suggesting that the fibrous avascular nature of the ovarian cortex could represent an inhospitable microenvironment for the dissemination of malignant cells. Nevertheless, the graft of ovarian tissue still represents an experimental and challenging technique, indicating that the use of oocytes should be currently considered as an easier and safer approach than ovarian tissue graft to restore fertility. Interestingly, immature oocytes could be easily retrieved from ovarian tissue during OTC in both prepubertal girls and women16, suggesting that it could represent a reliable source to maximize the fertility-restoring potential in addition to ovarian cortical tissue freezing. These oocytes could be aspirated manually ex vivo in the ART laboratory from visible antral follicles or be isolated from spent media after ovarian tissue dissection. Retrieved oocytes could then be directly vitrified at an immature stage or be matured before the vitrification step using in vitro maturation (IVM)20,21.
In this manuscript, we propose a protocol that combines the cryopreservation of ovarian tissue with the isolation and cryopreservation of mature (MII-stage oocytes) and/or immature oocytes (i.e., Germinal Vesicle (GV) and Metaphase I (MI)-stage oocytes) retrieved from ovarian tissue. This protocol outlines all the specific steps required to maximize the fertility preservation potential in both prepubertal girls and women.
All women (over 18 years of age) as well as all minor girls and their parents signed an informed consent form to preserve the fertility of the patient at risk of POI. This protocol is considered as a routine ART procedure in our center for fertility preservation. It follows the guidelines of our institutions human research ethics committee.
1. Quality control
2. The day before the preservation
3. Ovarian tissue collection and transport
Figure 1: Ovarian tissue cryopreservation. Ovarian tissue cryopreservation in a prepubertal girl (A, 7-year-old patient) and in a woman (B, 28-year-old patient) both suffering from acute myeloid leukemia. (A1–B1) Laparoscopic view of the ovary. (A2–B2–B3) Dissection of ovarian tissue. (B4) Cortical ovarian tissue. CL: corpus luteum. SAF: small antral follicle. Please click here to view a larger version of this figure.
4. Ovarian tissue preparation
5. Manual follicle aspiration from ovarian tissue samples
6. Ovarian tissue dissection
7. Manual COC isolation from the dissection spent media
Figure 2: Mature and immature COCs retrieved from ovarian tissue. Mature (A) and immature (B1–B3,C1–C2) COCs retrieved from ovarian tissue. (A) Mature COC containing a mature oocyte (presence of the first polar body, Metaphase II-stage oocyte). (B1) Healthy immature COC containing an immature oocyte (no polar body, Metaphase I-stage oocyte). (B2–B3) Healthy immature COCs containing immature Germinal Vesicle-stage oocytes (no polar body, Prophase I-stage oocytes). (C1–C2) Unhealthy COCs containing an atretic brown colored oocyte. Please click here to view a larger version of this figure.
8. Ovarian tissue histological analysis
9. Oocyte denudation and selection
Figure 3: Mature and Immature oocytes retrieved from ovarian tissue. (A) Mature oocyte (Metaphase II-stage oocyte: presence of the first polar body (PB, arrow), no visible nucleus), (B1–B2) Immature oocytes (Metaphase I-stage oocyte: no polar body, no visible nucleus), (C1–C3) Immature oocytes (Germinal Vesicle-stage (VG) oocytes: no polar body, presence of a large halo with nucleoli within the cytoplasm (arrow)). In C1, Germinal Vesicle-stage oocytes display heterogenous size. (D1–D3) Atretic oocytes. PB: polar body. GV: Germinal Vesicle. Please click here to view a larger version of this figure.
10. Oocyte vitrification
A total of 81 OTC from 81 female patients have been performed between 2007 and 2020, including 43 prepubertal girls and 38 women. The mean age of female patients (prepubertal girls and women) was 14.21 ± 9.61 years (mean ± standard error). The youngest patient was 5 months old and the oldest was 33.6 years old (Table 1). The mean age of prepubertal girls and women was 6.84 ± 4.81 years old (min–max: 0.5–15.1) and 22.76 ± 5.92 years old (min–max: 14.2–33.6), res...
The current manuscript provides a protocol combining ovarian tissue freezing and cryopreservation of oocytes retrieved from ovarian tissue, increasing the potential of fertility in prepubertal girls and women at risk of POI. We strongly recommend performing this protocol before the start of any gonadotoxic therapy in order to optimize the quantity (i.e., number of viable oocytes) as well as the quality (i.e., DNA integrity and cytoplasm competence) of the preserved oocytes, optimizing their safety for clinical use
None of the authors have competing interests.
We thank all members of our centers involved in the activity of fertility preservation (Gynecologists, Biologists, Oncologists, and Anatomopathologists). The study was conducted as part of the routine procedures for fertility preservation. No funding was received.
Name | Company | Catalog Number | Comments |
1 ml disposable syringe | CDD | 1323101/7002655 | Other material and sizes may also be suitable |
21-gauge syringe needle | Merck | Z192481 | Other material and sizes may also be suitable |
35 mm IVF Petri Dish | Nunc | 150255 | Other material and sizes may also be suitable |
60 mm IVF Petri Dish | Nunc | 150270 | Other material and sizes may also be suitable |
90 mm IVF Petri Dish | Nunc | 150360 | Other material and sizes may also be suitable |
Atraumatic forceps | Medlane | PI 299 04 | Other material and sizes may also be suitable |
Continuous Single Culture Complete with HSA | Irvine Scientific | 90165 | IVF culture medium for follicular fluid collection, COCs incubation, oocyte denudation and oocyte incubation until the vitrification step. |
Cryotube | Thermo Scientific | 368632 | Other products may also be suitable |
Dimethylsulfoxide (DMSO) | MILTENYI BIOTEC SAS | 170-076-303 | CryoMACS DMSO 10 (EP) |
GT40 | Air Liquide | 1,13,517 | Storage tank |
HSV High Security Vitrification Straw | Irvine Scientific | 25251 | Vitrification straws |
Human serum albumin | Vitrolife | 10064 | Other products may also be suitable |
Leibovitz L15 medium | Eurobio | CM1L15000U | Culture medium for ovarian tissue collection, transport and tissue dissection |
Leibovitz L15 medium | Eurobio | CM1L15000U | Culture medium for freezing solution |
Mars-IVF Class II Workstation/L126 IVF Dual | CooperSurgical | WM1500/6-133-911-121 | Workstation |
Programmable freezer | Planner KRYO 500 | Kryo 560-16 | Other equipements may also be suitable |
Scissors with sharp straight blades and finely sharpened points | Medlane | CI 034 03 | Other material and sizes may also be suitable |
Stripper | CooperSurgical | MXL3-STR-CGR | Other products may also be suitable |
Tips (150µm) for Stripper | CooperSurgical | MXL3-150 | Other products may also be suitable |
Vitrification Kit | Irvine Scientific | 90133 | Protocols are available at http://www.irvinesci.com/products/90133-so-vitrification-freeze-solutions. Other products may also be suitable |
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