Dr Lodovico Parmegiani
GynePro Medical Centers
Reproductive Medicine Unit
Via T. Cremona 8
+39 (0)347 472 5674
Efficiency of aseptic open vitrification in ultraviolet-sterilised Liquid Nitrogen and hermetical cryostorage of human oocytes: a prospective randomised study between fresh versus vitrified/warmed sibling oocytes.
The objective of the present study is to demonstrate that ultraviolet (UV) sterilisation of Liquid Nitrogen (LN2) is not detrimental and can be safely used for aseptic vitrification of human oocytes. In addition, in this study, we also describe a system to avoid hypothetical contamination during cryostorage, by enclosing the vitrified oocytes in a sterile device for hermetic isolation inside cryobanks. With this article we wish to report for the first time a safe method for aseptic open vitrification and hermetical cryostorage of human oocytes in accordance with the European directives on tissue manipulation. In order to assess the safety of this kind of vitrification/storage procedure we performed a randomised comparison between fresh and vitrified/warmed sibling-oocytes on infertile couples coming to our centre for intracytoplasmic sperm injection (ICSI) treatment.
The institutional medical ethics committee of GynePro Medical Center approved on the 16th of March 2008
Single centre prospective randomised study
Primary study design
Secondary study design
Randomised controlled trial
Patient information sheet
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Intracytoplasmic sperm injection (ICSI), aseptic open oocyte vitrification in UV-sterilised liquid nitrogen, hermetical cryostorage
After retrieval oocytes were denuded and then evaluated to assess their nuclear maturation stage. The oocytes that had released the first polar body (metaphase II - MII) underwent a strict selection by morphological features (zona pellucida thickness, perivitelline space size, oocyte shape, cytoplasm colour and granularity, presence of vacuoles and first polar body morphology). Immediately after decumulation and quality evaluation, the high quality MII oocytes were put in progressively-numbered culture droplets and randomised for ICSI; the supernumerary sibling MII oocytes were vitrified.Since at the beginning of this study the Italian IVF law allowed the injection of maximum three oocytes (Benagiano and Gianaroli, 2004), between April 2008 and 8 April2009 three MII oocytes were randomised for ICSI and the supernumerary sibling oocytes were vitrified. Randomisation was performed by a different embryologist to the operator who performed oocyte denudation using a specific software (www.randomizer.org). Since 9 April 2009 - due to changes in the Italian law (Benagiano and Gianaroli, 2010) - the number of oocytes to randomise for ICSI or vitrification has been defined following our centres guideline based on: female age at oocyte recovery and semen parameters, but generally not more than 6 oocytes are injected.
Only the first warming cycle per patient was included in the study: from the beginning of the study to 8 April 2009 maximum 3 random warmed oocytes were injected by ICSI; from 9 April 2009 the number of warmed oocytes to inject has defined following our centres guideline.
LN2 sterilisation via UV irradiation was performed by administration of 660,000 µW/cm2.
Cryotop (Kitazato BioPharma Co, Fuji-Shizuoka, Japan) oocyte vitrification was performed at room temperature in a 'home made' solution comprising 15% dimethylsulphoxide (DMSO- D 2438 Sigma Aldrich, Steinheim, Germany), 15% ethylene glycol (EG 10.246-6 Sigma Aldrich) and 0.5 mol/L sucrose (Sigma Aldrich), after a gradual initial equilibration of 15 minutes in a solution comprising 7.5% DMSO and 7.5% EG (Kuwayama et al, 2005, Rienzi et al., 2009). For the ultra-rapid cooling, the Cryotops - containing 1-2 oocytes- were plunged into UV-sterilised LN2 . and closed with their plastic caps. Then, the Cryotops of each patient were enclosed in 'home made' hermetical aluminium cylindric containers (high security goblets). which can contain up to 6 Cryotops each. These goblets had been previously submerged vertically in LN2 in order to avoid the infiltration of LN2 and checked for an inner temperature of -196° C at the end of UV-sterilisation process. The Cryotops were inserted into the 'high security goblets' taking care to keep the Cryotop strip containing the oocyte in the nitrogen vapour phase above the LN2. Finally, the goblets were hermetically closed with sterilised caps and polipropilene adhesive tape (Scotch® 3M Italia, Pioltello MI, Italy).
Before the rapid warming, the hermetical goblets containing the Cryotops were opened into the UV-sterilised LN2. The caps of the Cryotops were removed in the LN2, and each open carrier was submerged in 1 mL of warming solution containing 1 M sucrose at 37° C. Then, the oocytes were incubated at room temperature for 3 minutes first in 0.5 M and subsequently in 0.25 M and finally washed for 4 minutes in basic medium (PBS D8662 Sigma Aldrich, supplemented with 20% EHSA Conception Tecnologies, San Diego CA, USA) before culture. Warmed oocytes were considered to have survived in absence of negative characteristics: dark or contracted ooplasm, vacuolization, cytoplasmic leakage, abnormal perivitelline space, cracked zona pellucida. After 1-2 hours post-warm culture the surviving oocytes were inseminated by ICSI.
Primary outcome measure
1. Oocyte fertilisation rate
2. Embryo cleavage rate
3. Top-quality embryo rate
Secondary outcome measures
No secondary outcome measures
Overall trial start date
Overall trial end date
Reason abandoned (if study stopped)
Participant inclusion criteria
1. Women enrolled in the vitrification programme at GynePro medical centre undergoing ICSI with ejaculated spermatozoa
2. Not older than 41 years
3. At least six mature-Methaphase II oocytes at retrieval
This study compares the outcome of 31 warmed ICSI cycles performed from January 2009 to May 2010 with the outcome of fresh sibling oocytes ICSI performed from April 2008 to March 2010
Target number of participants
Participant exclusion criteria
1. Women older than 41 years old
2. Women with less than six mature-MII oocytes at retrieval
3. Patients with male partners with testicular spermatozoa or severe oligoastenoteratozoospermia (motile sperm count ≤ 500.000/mL after sperm preparation)
Recruitment start date
Recruitment end date
Countries of recruitment
Trial participating centre
GynePro Medical Centers
GynePro Medical Centers (Italy)
Funding Body Type
Funding Body Subtype
Results and Publications
Publication and dissemination plan
Not provided at time of registration
Intention to publish date
Participant level data
Not provided at time of registration
Basic results (scientific)
1. 2011 results in http://www.ncbi.nlm.nih.gov/pubmed/21843968
Parmegiani L, Cognigni GE, Bernardi S, Cuomo S, Ciampaglia W, Infante FE, Tabarelli de Fatis C, Arnone A, Maccarini AM, Filicori M, Efficiency of aseptic open vitrification and hermetical cryostorage of human oocytes., Reprod. Biomed. Online, 2011, 23, 4, 505-512, doi: 10.1016/j.rbmo.2011.07.003.