Fertility Preservation Through Oocyte Vitrification: Clinical and Laboratory Perspectives

Summary

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.

Abstract

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.

Introduction

The development and implementation of an efficient cryopreservation program for human oocytes has been a significant breakthrough in reproductive medicine. According to recent evidence, vitrification is the most effective strategy to cryopreserve metaphase II (MII) oocytes, as it results in statistically higher survival rates compared to slow freezing, independently of the patient population (infertile patients or oocyte donation program)^1,2,3. The remarkable achievements of oocyte vitrification led the Practice Committees of the American Society for Reproductive Medicine (AS....

Protocol

1. Work-up and clinical counseling

NOTE: In case of patients requiring fertility preservation for oncologic reasons, ensure that there is no waiting list for scheduling consultation, and the appointment is provided as soon as possible.

1. Examine the medical history and previous documentation, and assess the patient's general health status.
2. Record all information (including the oncologist's approval to undergo ovarian stimulation in case of cancer patients) in a relational database.
3. Provide the patient with specific counseling about the feasibility of the procedure. Explain the steps of the procedure (ov....

Results

Overview of the fertility preservation program at the center

Over a 12-year period (2008-2020), 285 women underwent at least one oocyte retrieval entailing the vitrification of the whole cohort of mature eggs collected. Most of these women (n=250) underwent a single retrieval, and 35 underwent multiple retrievals. The reasons for undergoing oocyte retrieval for egg vitrification are summarized into 4 categories: medical (except for cancer), cancer, non-medical, and others. Amo.......

Discussion

Clinical considerations

Although emerging strategies, such as ovarian tissue cryopreservation and in vitro maturation, have been explored, oocyte vitrification after COS is the gold standard technique for fertility preservation. In this scenario, the number of oocytes retrieved and cryopreserved should be maximized in the shortest possible time as most cancer patients might benefit solely from one ovarian cycle before they have to commence their cancer treatment(s). Thus, a p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collection
Equipment
Hot plate	IVF TECH
Lab Markers	Sigma Aldrich
Laminar Flow Hood	IVF TECH		Grade A air flow
Stereomicroscope	Leica	Leica M80
Thermometer
Test tube Warmer
Tri-gas incubator	Panasonic	MCO-5M-PE	02/CO2
Vacuum Pump	Cook	K-MAR-5200
Consumables
CSCM (Continuos single culture complete) medium	Fujifilm Irvine Scientific	90165	IVF culture medium supplemented with HSA
Mineral Oil for embryo culture	Fujifilm Irvine Scientific	9305
Ovum Aspiration Needle (Single lumen)	Cook	K-OSN-1730-B-60
Primaria Dish	Corning	353803	Corning Primaria Dish 100x20 mm style standard cell culture dish
Round- bottom tubes	Falcon	352001	Falcon 14ml Round Bottom Polystyrene Test tube with snap cap
Round- bottom tubes	Falcon	352003	Oocyte collection tubes/ Falcon 5ml 12x75 Round Bottom Polipropilene Test tube with snap cap
Rubber Bulb	Sigma Aldrich	Z111589-12EA
Sterile glass Pasteur pipettes	Hunter Scientific	PPB150-100PL	Pipette Pasteur Cotonate, 150mm, MEA e CE
Denudation
Equipment
CO2 incubator	Eppendorf	Galaxy 14S
Flexipet adjustable handle set	Cook	G18674	Stripper  holder
Gilson Pipetman	Gilson	66003	p20
k-System Incubator	Coopersurgical	G210Invicell
Lab Markers	Sigma Aldrich
Laminar Flow Hood	IVF TECH		Grade A air flow
Stereomicroscope	Leica	Leica M80
Consumables
Biopur epTIPS Rack	Eppendorf	30075331	Micropipettes epTIPS Biopur 2-200 µl
Human Serum Albumin	thermoFisher Scietific	9988
Hyaluronidase	Fujifilm Irvine Scientific	90101	80 IU/mL of hyaluronidase enzyme in HEPES-buffered HTF
IVF culture dish (60 x 15mm)	Corning	353802	Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish
IVF dish 4-well plate with sliding lid	ThermoFisher Scietific	176740	Multidishes 4 wells (Nunc)
IVF One well dish	Falcon	353653	Falcon 60 x 15 mm TC treated center-well IVF
Mineral Oil for embryo culture	Fujifilm Irvine Scientific	9305
Modified HTF Medium	Fujifilm Irvine Scientific	90126	HEPES-Buffered medium
Rubber Bulb	Sigma Aldrich	Z111589-12EA	1 mL for pasteur pipettes
Sterile glass Pasteur pipettes	Hunter Scientific	PPB150-100PL	Pipette Pasteur Cotonate, 150 mm, MEA e CE
stripping pipette  tips (140 µm)	Cook	K-FPIP-1140-10BS-6	PIPETTE FLEXIPETS PER DENUDING
stripping pipette tips (130 µm )	Cook	K-FPIP-1130-10BS-7	PIPETTE FLEXIPETS PER DENUDING
stripping pipette tips (170 µm)	Cook	K-FPIP-1170-10BS-5	PIPETTE FLEXIPETS PER DENUDING
Vitrification
Equipment
Electronic Timer
Flexipet adjustable handle set	Cook	G18674	Stripper  holder
Gilson Pipetman	Gilson	F123601	p200
Lab Markers	Sigma Aldrich
Laminar Flow Hood	IVF TECH		Grade A air flow
Stainless Container for Cooling Rack	Kitazato		Liquid nitrogen container for vitrification
Stereomicroscope	Leica	Leica M80
Consumables
Biopur epTIPS Rack	Eppendorf	30075331	Micropipettes epTIPS Biopur 2-200 µL
Human Serum Albumin	Fujifilm Irvine Scientific	9988
IVF culture dish (60 x 15 mm)	Corning	353802	Corning Primaria Falcon Dish 60 x 15 mm TC Primaria standard cell culture dish
IVF dish 6-well	Oosafe	OOPW-SW02	OOSAFE 6 WELL DISH WITH STRAW HOLDER
Modified HTF Medium	Fujifilm Irvine Scientific	90126	HEPES-Buffered medium
stripping pipette tips (170 µm)	Cook	K-FPIP-1170-10BS-5	PIPETTE FLEXIPETS PER DENUDING
Vitrification Freeze kit	Fujifilm Irvine Scientific	90133-so	2 Vials of ES (Equilibration Solution, 2 x 1 mL) and 2 Vials of VS (Vitrification Solution, 2 x 1 mL)
Vitrifit	Coopersurgical Origio	42782001A	VitriFit  Box
Warming
Equipment
Electronic Timer
Flexipet adjustable handle set	Cook	G18674	Stripper  holder
Gilson Pipetman	Gilson	F123601	p200
k-System Incubator	Coopersurgical	G210Invicell
Lab Markers	Sigma Aldrich
Laminar Flow Hood	IVF TECH		Grade A air flow
Stainless Container for Cooling Rack	Kitazato		Liquid nitrogen container for vitrification
Stereomicroscope	Leica	Leica M80
Consumables
Biopur epTIPS Rack	Eppendorf	30075331	Micropipettes epTIPS Biopur 2-200 µL
CSCM (Continuos single culture complete) medium	Fujifilm Irvine Scientific	90165	IVF culture medium supplemented with HSA
IVF culture dish (60 x 15 mm)	Corning	353802	Corning Primaria Falcon Dish 60X15mm TC Primaria standard cell culture dish
IVF dish 4-well plate with sliding lid	ThermoFisher Scietific	176740	Multidishes 4 wells (Nunc)
IVF dish 6-well	Oosafe	OOPW-SW02	OOSAFE® 6 WELL DISH WITH STRAW HOLDER
Mineral Oil for embryo culture	Fujifilm Irvine Scientific	9305
SAtripping pipette tips (300µm)	Cook	K-FPIP-1300-10BS-5	PIPETTE FLEXIPETS PER DENUDING
Vitrification Thaw kit	Fujifilm Irvine Scientific	90137-so	4 Vials of TS (Thawing Solution, 4 x 2 mL) + 1 Vial of DS (Dilution Solution, 1 x 2 mL) +1 Vial of WS (Washing Solution, 1 x 2 mL)