Safety and efficiency of oocyte vitrification

Accepted Manuscript Safety and efficiency of oocyte vitrification Neelke De Munck, Gábor Vajta PII:

S0011-2240(17)30100-1

DOI:

10.1016/j.cryobiol.2017.07.009

Reference:

YCRYO 3871

To appear in:

Cryobiology

Received Date: 20 March 2017 Revised Date:

25 July 2017

Accepted Date: 25 July 2017

Please cite this article as: N. De Munck, Gá. Vajta, Safety and efficiency of oocyte vitrification, Cryobiology (2017), doi: 10.1016/j.cryobiol.2017.07.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Abstract As the oocyte is the starting point for a new life, artificial reproductive technology (ART) techniques should not affect the (ultra) structural and functional integrity, or the developmental competence. Oocyte vitrification -one of the most significant achievements in human ART during the past decadeshould therefore be a safe and efficient technique. This review discusses the principles and developments of the existing and future techniques, applications possibilities and safety concerns.

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The broad range of vitrification media and devices that are currently available, show differences in their effects on the oocyte ultrastructure and preimplantation development. It is not yet fully decided whether this has an influence on the obstetric and neonatal outcome, since only limited information is available with different media and devices. For autologous oocytes, the obstetric and neonatal outcomes appear promising and comparable to pregnancies obtained with fresh oocytes. This however, is not the case for heterologous fresh or vitrified oocytes, where the immunological foreign foetus induces adverse obstetric and neonatal outcomes. Besides the oocyte vitrification process itself, the effect of multiple stimulations (for oocyte banking or for oocyte donors), seems to influence the possibility to develop gynaecological cancers further in life.

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Automated vitrification/warming should offer a consistent, cross-contamination free process that offers the highest safety level for the users. They should also produce more consistent results in survival, development and clinical pregnancies between different IVF clinics.

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ACCEPTED MANUSCRIPT 1

Safety and Efficiency of Oocyte Vitrification.

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De Munck, Neelke; Vajta, Gábor

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Introduction Widespread use of vitrification for cryopreservation of oocytes and embryos is one of

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the most significant achievements in human ART during the past decade. Although often

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called as "new" technology, vitrification has been applied successfully for mammalian

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embryos 33 years ago [76]. The delay in routine application can be explained by the slow

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advancement in optimization of parameters, as well as the aversion of professionals to use an

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experimental procedure for valuable human reproductive cells.

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watershed that happened approximately ten years ago, commonly used vitrification

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techniques are still extremely primitive ones, based entirely on the simple manual work of

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operators. Although the outcome justifies the application, there is an increasing demand for

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more sophisticated procedures that may eliminate potential dangers and inconsistencies

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related to human factors. The purpose of this review is to summarize the principles and the

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development of the existing techniques, to discuss various application possibilities and safety

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concerns. Finally, an attempt is made to outline the route for elimination of these concerns

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and to introduce new approaches that meet the level of the 21st century technology and the

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demand of modern assisted reproduction in humans.

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Vitrification

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In fact, in spite of the

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In contrast to traditional slow-rate freezing, where a delicate balance is maintained

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through the induction of extra-cellular ice crystals, vitrification focuses on the total

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elimination of ice crystal formation in both the extra- and intracellular solutions. In

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embryology, this goal is usually achieved by transferring the oocytes to a solution with a

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relatively high concentration of cryoprotectants, as well as by using extremely high cooling

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and warming rates and by using small (<1µl) solution volumes that are exposed directly to

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liquid nitrogen. The small volume also prevents heterogenous ice crystal formation, and the

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high cooling and warming rate at relatively high temperatures decreases chilling injuries.

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In spite of the extensive basic research that explains in detail events during

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vitrification, all steps in the development of the procedure have happened empirically,

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including the selection of the most efficient cryoprotectant mixture, development of the

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ACCEPTED MANUSCRIPT proper carrier tool, finding the right final cryoprotectant concentration and outlining

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parameters of optimal equilibration. For permeable cryoprotectants, equal proportions of

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dimethylsuphoxide and ethylene glycol were found highly efficient and reliable [43].

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Concerns related to the toxicity of the former component were mostly based on

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misunderstandings and were not justified by the outcome; moreover, the suggested alternative

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(propylene glycol) was proven to be more toxic by various investigations [5, 97]. For non-

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permeable cryoprotectants, no convincing evidence is available to prove the superiority of

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either sucrose or trehalose, both sugars are used widely in solutions for cooling and warming.

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Development of the carrier tool was retrospectively a simple task, however, the discovery of

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the first model in bovine oocytes [56] and the first purpose-made designs required almost ten

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years [41,48,93]. Although new carrier tools are introduced almost every year, none of them

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was/is found essentially superior to these first devices. The minimal volume - direct contact

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to liquid nitrogen has helped to decrease the required concentration of permeable

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cryoprotectants by 25-30% [56,93]. However, the adjustment of the optimal multistep

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equilibration parameters, required further years, almost a decade [48,72].

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Retrospectively the key of the success was (i) to reach a full equilibrium with a

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relatively low concentration (7.5% for both components) of permeable cryoprotectants with a

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relatively long (possibly multistep) exposure time at relatively low (25°C) temperature, to

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avoid osmotic and toxic injuries, respectively; (ii) to make a short and aggressive

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compression-dehydration of the cells with exposure to a higher concentration (15-17% for

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both components) of permeable cryoprotectants mixed with a concentrated (0.7 to 1M) non-

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permeable cryoprotectant and (iii) loading the sample to the carrier tool and immersion into

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liquid nitrogen - or analogue cooling agent.

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Among various agents tested for additional protection, human serum derivates and

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recently a semisynthetic plant derivate, hydroxypropyl-methylcellulose [62] were found

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useful, most probably by protecting membranes during the cryopreservation process. On the

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other hand, cytoskeleton relaxants and antifreeze proteins were eventually found of little or

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no use [31, 82]. Attempts to further increase cooling rates by using liquid nitrogen

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supercooled in vacuum, or helium were found unpractical and their application was not

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justified by the overall outcome [85].

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Warming is usually performed by direct immersion of the sample in pre-heated

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solutions (37°C) to ensure the highest warming rate. The solution contains a highly

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concentrated

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Subsequently, a slow and careful decrease of the cryoprotectant concentration is applied to

(0.5-1M)

non-permeable

cryoprotectant

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avoid

osmotic

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ACCEPTED MANUSCRIPT ensure a mild rehydration without causing osmotic damage in membranes that are rather

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fragile as a consequence of the cryopreservation. By applying the complete procedure correct,

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close to 100% survival rates should be obtained with preservation of the developmental

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competence comparable to that of fresh human oocytes.

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The oocyte and the developing embryo

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The mature human oocyte has a specific chromatin and cytoplasmic arrangement that is

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important to achieve fertilization and adequate development [25, 90].

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During the process of vitrification, the oocyte is exposed to numerous physical and chemical

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processes that fluctuate over a wide non-physiological range which may impact structural and

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genomic integrity [47]. Since the oocyte -as a single cell structure- is the starting point for a

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new life, the repeated volumetric changes during vitrification and warming should not affect

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the oocyte structure and further development.

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While in the beginning of oocyte vitrification most efforts were made to increase efficiency

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in terms of survival, it becomes clearer that efficiency should coincide with safety [9,40].

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Even if safety is interpreted in terms of the health of liveborns, it should also include the

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structural modifications at the oocyte level and evaluate their biological impact.

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Unfortunately, these safety studies are rather scarce and not yet determined for the wide

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range of vitrification devices and protocols available to date (Table 1). One of the

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ultrastructural changes observed after oocyte vitrification is the appearance of vacuoles -most

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probably caused by swelling and coalescence of isolated smooth endoplasmic reticulum

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(SER) vesicles- which are responsible for the inward organelle replacement [38]. As

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microfilaments arrange the organelles throughout the oocyte in a time-dependent manner, this

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phenomenon might have negative developmental consequences. This vacuolization appears

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to be more common in closed than in open vitrification devices [8]. Misalignment of

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chromosomes -and ultimately aneuploidy- may arise due to microtubular network disruptions

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that displace the spindle [89]. A reduction in the fertilization potential can be caused by the

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abnormal distribution of the mitochondria or changes in the mitochondria-SER complexes

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that disturb the Ca2+ homeostasis. The effect of cryopreservation on the epigenetic marks in

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different animal models was recently reviewed by Chatterjee et al. [14]. The only two studies

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on human oocytes did not report aberrant epigenetic changes so far [1, 30].

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Different oocyte sources may exist, all with a different inherent quality that affect the

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vitrification safety and/or efficiency. Young donor oocytes are expected to be of good

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ACCEPTED MANUSCRIPT quality. Patients vitrifying oocytes in the prevention of age-related fertility decline should do

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this before the age of 36, since after this age, the survival and live birth rate decreases [28].

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Infertile patients show less favourable clinical pregnancies per warmed oocyte when

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compared to fresh oocytes [75]. IVM and ex vivo IVM (IVM on immature oocytes retrieved

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from the extracorporeal ovarian tissue after ovariectomy) oocytes are thought to be of a lower

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quality due to a lower concentration of mitochondrial DNA, swollen mitochondria and an

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abnormal spindle and chromosome configuration [54]. The effect of oocyte vitrification on

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the embryo development is presented in Table 2.

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Safety

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Safety is “the state of being protected from danger or harm, or the condition of not being

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likely to cause damage or harm” [10].

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For oocyte vitrification, safety means that an embryo transfer from vitrified oocytes results in

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an uncomplicated delivery of a healthy child.

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Safety implies that all steps in an oocyte vitrification/warming cycle should be validated as

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safe. During recent years, a lot of effort was made to determine the safety of ART techniques

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and when/how new technologies should be introduced in an IVF lab [9,40]. For oocyte

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vitrification, however, the basic research and follow-up studies (first safety assessment steps)

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were performed only after its widespread clinical application [40]. The first case report on

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human oocyte vitrification combined a clinical application with very little basic research [49].

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In this report, oocytes were vitrified with the use of the Open Pulled Straw (OPS) especially

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designed to generate very high cooling and warming rates [94]. This led to a tremendous

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increase in publications of clinical studies and was only slowly followed by more basic

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research on vitrified oocytes [19, 20, 22, 48, 49].

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Obstetric safety

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Even though the clinical application of oocyte vitrification only started a decade ago, already

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thousands of children are born after this technique; most of them after oocyte donation.

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Depending on the oocyte source being used, heterologous or autologous, different obstetric

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outcomes are to be expected.

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ACCEPTED MANUSCRIPT Heterologous oocytes

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Only a limited number of publications (prospective and observational studies) is available

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analysing the obstetric outcome after oocyte vitrification. Most of them after vitrification

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with an open device [16,22,23,67] and only one with a closed device [28]. For the open

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vitrification device, the largest series (fresh: 516 singletons and 160 multiple pregnancies;

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vitrified: 503 singletons and 201 multiple pregnancies) was analysed by Cobo et al. [23]

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where fresh and vitrified donor oocyte cycles were compared. The incidence of pregnancy-

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induced hypertension was 15.1% (vitrified: 11.7%) for singleton and 18.8% (vitrified: 18.9%)

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for twin pregnancies. First trimester vaginal bleeding was observed in 28.5% (vitrified:

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32.8%) and 37.9% (vitrified: 40.4%), respectively and the overall bleeding was 33.3%

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(vitrified: 39.6%) and 47.3% (vitrified: 52.7%). A relative high twinning rate of 28.6% was

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obtained in this study. The observational cohort study with the closed device, tough on a

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much lower number of pregnancies (95 singleton and 22 twin pregnancies), showed a high

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prevalence of hypertensive disorders (19.6%) and haemorrhages (26.8%) and a twin

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pregnancy rate of 18.8% [28].

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Patients appealing for donor oocytes belong to a heterogeneous population. Advanced

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maternal age (AMA), premature ovarian failure, low ovarian reserve, genetic conditions and

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multiple IVF failures are different indications for the use of donor oocytes. AMA represents

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the largest group in this population and is known to be associated with adverse pregnancy

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outcomes like hypertensive disorders, gestational diabetes and preterm labour [46,87]. A

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recent meta-analysis on pregnancy complications after oocyte donation concluded that oocyte

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donation in se is an independent risk factor for obstetric complications [44]. This indicates

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that the use of vitrified oocytes, irrespective of the vitrification device, does not increase the

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obstetric risks when compared to fresh donor oocytes. The adverse outcomes might be

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explained by the impaired placentation of the immunologically foreign foetus compared with

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pregnancies obtained with autologous oocytes (semi-allograft). During invasion, it is

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important that the mother (gestational carrier) is not rejecting the allogeneic foetus; this is

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obtained by complex mechanisms of immunoregulation [58]. When compared to non-donor

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IVF pregnancies, placental analysis after oocyte donation showed an increased

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immunological activity at the maternal-foetal interface that could either represent a host

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versus graft rejection-like phenomenon or an effort to suppress rejection [39]. The fact that

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adverse outcomes decrease when the oocyte donor is related to the recipient suggests a lower

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ACCEPTED MANUSCRIPT HLA mismatch between relatives. A reduction in adverse outcomes is believed to be obtained

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by a higher HLA match between donor and recipient [83].

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Twin pregnancies are not a direct consequence of the oocyte donation, but the result of the

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transfer of more than one embryo. Since young donor oocytes are used for transfer, single

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embryo transfer should be the way to go in first and second attempts.

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Autologous oocytes

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Depending on the indication and the age at which the vitrification is performed, different

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outcomes are to be expected. The largest analysis of the obstetric outcome for non-medical

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indications after oocyte vitrification only found a higher number of caesarian sections when

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compared to fresh oocytes for the singleton pregnancies (n=81), and no difference for the

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twin pregnancies (n=19) [23]. For the oncological indications, after ovarian stimulation, after

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in vitro maturation (IVM) or after ex vivo IVM, it is yet too early to comment on any effect of

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vitrification on the obstetric outcome.

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Neonatal safety

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Heterologous oocytes

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The meta-analysis by Jeve et al. [44] showed that fresh oocyte donation is associated with an

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increased risk for the birth of a child that is small for gestational age (SGA). This finding is

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not abnormal, since these authors also found that oocyte donation was associated with

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hypertensive disorders and preeclampsia due to a defective placentation. These conditions

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will often lead to intrauterine growth restriction and preterm delivery leading to SGA. The

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reports on neonatal outcome after open or closed oocyte vitrification in oocyte donation

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programmes appear promising [23,23]. As with the obstetric outcome, the neonatal outcome

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is affected by the oocyte source (donor in this case) and not by the way the oocyte is handled

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(fresh, open vitrification, closed vitrification).

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Autologous oocytes

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Data about the neonatal outcome obtained with vitrified autologous oocytes is very scarce.

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The analysis by Cobo et al. [23], comparing fresh and vitrified oocytes, showed a higher

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incidence of minor malformations (OR=13.0; p<0.2) after oocyte vitrification for the 81

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singleton pregnancies compared to the 252 singleton pregnancies with fresh oocytes. No

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ACCEPTED MANUSCRIPT neonatal differences were observed between the 68 fresh and the 19 vitrified twin

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pregnancies. Larger numbers are needed to define whether or not oocyte vitrification

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influences the neonatal outcome.

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Donor safety

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The use of donor oocyte banks reduced the pre-existing logistic problems like donor-recipient

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synchronization or the allocation of oocytes to different recipients. Unfortunately, little

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information is provided on the effect of the multiple stimulations on the donors. Instead of

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generating one mature oocyte per cycle (by single dominant follicle selection) high doses of

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exogenous gonadotrophins are administered to stimulate the development of multiple oocytes

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to mature in a single cycle [84]. This leads to the production of supraphysiological serum

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oestradiol levels (E2). In a normal menstrual cycle, E2 levels of 300pg/mL are observed,

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while highly stimulated cycles may peak up to 4000pg/mL. It has been shown that longer

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lifetime exposure to endogenous estrogen is associated with an increased risk of breast cancer

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[42].

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development of ovarian cancer [45].

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The use of fertility medications and its effect on the development of cancer was recently

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reviewed [50]. Except for the fact that infertile patients are at increased risk for the

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development of gynaecological cancers, no clear significant risk was found with the use of

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fertility medications. A recent publication on the health after oocyte donations concluded that

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oocyte donation was not associated with harmful long-term general or reproductive health

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effects [88]. There were no reports on ovarian or uterine cancer and only one report of breast

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cancer in this study. However, a large proportion of the analysed donors were still very

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young; the analysis was performed in 2013 and donation cycles until 2012 were included and

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thus the long-term effects could not be described for this population. Also, the donors with

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donation cycles between 2008 and 2012 had a higher proportion of young donors (<24 years)

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and a higher proportion of childless donors at the time of donation. Analysis of these donors

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in 20 years could provide more accurate results on the long term effects of young childless

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donors.

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LN2-mediated disease transmission

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Among diverse concerns about safety of vitrification, the liquid nitrogen-mediated disease

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transmission was the most disputed issue [7]. Theoretically, there are arguments that support

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ACCEPTED MANUSCRIPT this possibility, as liquid nitrogen is difficult to sterilize and especially to keep it sterile when

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used for sample storage. There were different attempts to eliminate or decrease the obvious

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danger of contamination, including the suggestion of "safe, closed" devices for both cooling

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and storage; to sterilize liquid nitrogen for cooling with open devices, then inserting the

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cooled sample into a precooled container that is subsequently sealed for storage in liquid

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nitrogen dewars [94]; or after cooling in sterile nitrogen, arranging storage in liquid nitrogen

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vapour containers [21]. However, up till today, most laboratories use open devices for both

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cooling and storage in liquid nitrogen.

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There are very few commercially available vitrification devices that completely meet the

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requirement of sterility [98]. The majority of reportedly "closed" devices are partially open,

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exposing samples to vapour of liquid nitrogen, or closed only during the storage, not in the

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cooling phase. Others make cooling and storage in hermetically closed containers, but expose

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the sample to infection during warming. Data reported with the only existing, really closed

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and safe device [99] are still limited and obtained only from a handful of laboratories

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[28,37,71]

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However, the main reason to remain tolerant towards the very popular and efficient

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techniques is that -in spite of millions of transfers performed with samples cryopreserved

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with open vitrification devices- not a single report has been published on liquid nitrogen-

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mediated disease transmission in embryology, qualifying this approach as exceptionally

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harmless among interventions performed in human medicine. Possible reasons of this

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somewhat surprising outcome were discussed in detail in a recent review [98], some of the

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arguments have been confirmed lately [59].

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Operator safety

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In contrast to the danger of disease transmission related to exposure to liquid nitrogen, the

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safety of operators working in vitrification in an embryo laboratory is rarely discussed. The

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controversial situation has been analysed in detail recently [97]. Hazards during any kind of

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work with liquid nitrogen are considerable, accordingly strict standards have been established

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and mostly enforced by authorities including the use of special cryogloves, safety goggles,

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heavy rubber boots, protective clothing and face masks. Unfortunately available techniques

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of vitrification simply cannot be performed by keeping all these safety measures. Delicate

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manual handling, frequent change between microscopic and macroscopic follow-ups are

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hampered by safety gloves and goggles. On the other hand, complete elimination of safety

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ACCEPTED MANUSCRIPT measures exposes inexperienced people to serious danger, and there are no established

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standards for their education. Unfortunately the introduction and widespread use of

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vitrification has increased the danger considerably, as it requires manual work with - mostly

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homemade - open containers filled with litres of liquid nitrogen.

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Also, neglecting safety concerns has already resulted in one death in one embryo unit [4].

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Efficiency

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Efficiency is “a situation in which a person, company, factory, etc. uses resources such as

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time, materials or labour well, without wasting any; or a situation in which a person, system,

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or machine works well and quickly, or the difference between the amount of energy that is put

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into a machine in the form of fuel, effort, etc. and the amount that comes out of it in the form

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of movement; or ways of wasting less time, money, labour, etc.” [10].

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Oocyte vitrification efficiency can be defined as the fastest way to a live birth with the lowest

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number of vitrified oocytes. It is clear that the efficiency differs between the oocyte source

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that is used. Most data on efficiency is available from young oocyte donors, infertile patients

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or patients preserving for anticipated gamete exhaustion. It is yet unclear whether these

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results can be extended to oocytes after IVM or ex-vivo IVM.

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The interplay between the cooling and the warming rate and their influence on oocyte survival, embryo development and pregnancy

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The cooling rate

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A decade ago, it was believed that the success of oocyte survival after vitrification was

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predominantly determined by the cooling rate. This belief was mainly based on the results of

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open and closed devices available at that time. Not only was the oocyte survival rate higher

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with the open devices but also clinical pregnancy rates per warmed oocyte appeared to be

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better than with the closed devices [8,19,20,48,69,74,77,78].

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Unfortunately, studies comparing open and closed devices are very scarce with conflicting

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results on survival, fertilization, developmental competence and clinical pregnancy

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[29,69,70].

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ACCEPTED MANUSCRIPT The warming rate

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The effect of different cooling and warming rates on oocyte survival after vitrification has

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been studied on mouse oocytes by Seki and Mazur [86] and Mazur and Seki [57]. The

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authors demonstrated the dominance of the warming rate over the cooling rate to obtain better

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oocyte survival. Oocytes cryopreserved with a very low cooling rate (<200°C/min) but with a

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high warming rate (>2,000°C/min) tend to survive better compared to oocytes that are cooled

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very rapidly and warmed slowly. From this point of view, oocytes cryopreserved with a

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closed vitrification device are able to survive if a very high warming rate is applied, despite

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the low cooling rate. Their hypothesis suggests that a high proportion of the observed cell

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loss is attributed to recrystallization above Td since a low warming rate allows more time for

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recrystallization. Td is the devitrification temperature: once a glass warms above the

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devitrification temperature, intracellular ice may form due to recrystallization. This

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temperature explains the need for high warming rates, reducing the chance on

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recrystallization. It should be noted that these experiments were performed in mouse oocytes

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-which are extremely tolerant to cryodamage- and that the developmental competence

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(fertilization/development) was not evaluated. The role of a high warming rate was

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demonstrated on human oocytes by comparing two warming rates in a closed vitrification

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device [26]. Oocytes warmed with the highest warming rate showed a higher survival rate

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(89.6% versus 65.9%). Furthermore, a significantly higher fertilization and embryo

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development up to day 6 were obtained with the use of a higher warming rate. This indicates

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that, even though the oocytes appear morphologically survived, lowering the warming rate

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may induce some ultrastructural changes, invisible with light microscopy, that impair further

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development.

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The use of a very high warming rate in the closed Safespeed device (200.000°C/min) showed

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high survival, fertilization and developmental rates (and pregnancy rates: 3/6) for human

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oocytes, again indicating the importance of a high warming rate [35].

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The cooling and warming rate: pre-implantation effects

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While open devices are able to generate very high cooling and warming rates (e.g. both

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>20,000°C/min for the OPS device and >20,000°C/min and 40,000°C/min for the Cryotop

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device, respectively), for the closed devices these values are much lower (e.g. 2,900°C/min

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and 25,000°C/min for the CBSvit device, respectively). If a cooling rate of 2,900°C/min is

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ACCEPTED MANUSCRIPT not sufficient to obtain complete vitrification, as has been questioned for closed devices [96],

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the formation of very small ice crystals during vitrification cannot be excluded. These ice

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crystals are not necessarily harmful during the vitrification process, but are able to

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recrystallize during warming due to the slightly lower warming rate. Apparently, this process

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is not immediately lethal for the oocyte, but might be lethal for later fertilization and embryo

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development. This is seen when oocytes are analysed during warming. Oocytes vitrified with

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an open device show a less extensive compression during dilution and show a faster re-

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expansion [8,32]. These major differences in compression and re-expansion indicate that the

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device itself (and the related cooling and warming rate) has a major impact on the oocyte’s

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behaviour upon warming. Keeping this in mind, one may question the biological competence

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of morphologically surviving oocytes. Besides the differences in compression, differences in

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the development up to day 3 have also been described when comparing open and closed

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devices [29]. It is still unclear whether the slightly impaired development is attributed to (i)

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the lower cooling rate whether or not combined with a reduced warming rate, (ii) the higher

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uptake of toxic cryoprotectant or (iii) a combination of both.

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The cooling and warming rate: post-implantation effects

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The clinical relevance of the differences between open and closed devices and their

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corresponding cooling and warming rate has not yet been fully examined. Only one

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prospective RCT is available comparing the clinical efficiency of open (Cryotop) and closed

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(Vitrisafe) oocyte vitrification [70]. In this study, a significantly higher survival rate (91.0%

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versus 82.9%) was obtained in the open Cryotop device but no differences in the live birth

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rate per cycle were observed (24.0% versus 36.0%). This makes the authors conclude that the

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replacement of the open vitrification device by a closed vitrification device has no impact on

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biological and clinical outcome rates. However, their results are not in line with other studies

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using open Cryotop vitrification [22,78]. With donor oocytes, an ongoing pregnancy rate (10-

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11 weeks after embryo transfer) per recipient (intended to treat) of 43.7% was obtained [22],

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which seems higher than the ongoing pregnancy rate (33.3% in the closed device and 24.0%

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in the open device) by Papatheodorou et al. [70]. However, the recipient population in the

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latter study is on average three years older than in the study by Cobo et al. [22], which may

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contribute to the lower implantation and clinical pregnancy rate. To be able to draw

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conclusions regarding the superiority of one device over the other, more and larger RCTs are

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needed that compare the delivery outcomes between open and closed devices [98].

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ACCEPTED MANUSCRIPT Fresh, vitrified or slow-frozen oocytes?

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The question can be divided into two parts. Oocyte cryopreservation is an option that may

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offer solution in various medical and non-medical situations, as discussed in detail in recent

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reviews [17,73,80]. Briefly, medical indications include risk of infertility because of

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gonadotoxic treatments including that of cancer and other medical conditions. With the

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successful application of luteal phase stimulation [92], the main argument to use ovarian

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cryopreservation in these cases -to avoid delays- has weakened. Most cases of oocyte

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donation may also be regarded as medical indications. Non-medical indications belong

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mostly to the so called "social freezing" group. This group is increasing rapidly: in some

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countries up to 30% of women may consider oocyte cryopreservation because lack of partner

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or postponement of fertility for professional or other reasons [73]. Oocyte cryopreservation

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may also be required in countries where embryo cryopreservation is banned, or in cases

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where couples have moral concerns regarding deep-temperature storage of embryos.

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Regarding the preferred choice of cryopreservation technique, i.e. vitrification vs. slow rate

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freezing, the rapidly increasing amount of clinical evidence has resulted in a sharp change

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during the past decade, especially in the past five years. The first detailed review has

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suggested a complete elimination of use of programmable freezing machines for oocytes and

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embryos [95] due the lack of basic scientific studies in vitrification. However, it was not the

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subsequent intensive academic research, but the exponentially increasing amount of clinical

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data that has convinced the vast majority of laboratories. After publication of hundreds of

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papers in the subject, recent reviews unanimously suggest the use of vitrification for

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cryopreservation of all stages of human oocyte and preimplantation embryo development

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[3,52,79].

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The most recent meta-analysis by Potdar et al. [75] investigated the outcomes after

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(autologous and heterologous) oocyte vitrification. The primary aim of this review was to

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provide information on oocyte survival and pregnancy outcomes to help woman making

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informed choices before losing their reproductive potential due to gonadotoxic treatment or to

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increasing age. In addition, the authors compared the outcomes obtained with fresh and

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vitrified donor oocytes. It appeared that fresh and vitrified oocytes perform equally well

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regarding fertilization rate (OR=0.96 [0.87,1.06]), ongoing pregnancy rate (OR= 1.10

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[0.87,1.40]) and clinical pregnancy rate (OR=1.01 [0.84,1.23]) per warmed oocyte. However,

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only four studies [19,22,36,91] were included in this analysis of which one had a very large

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sample size [22]. A recent publication using the closed Vitrisafe device found no difference

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ACCEPTED MANUSCRIPT 375

in clinical pregnancy rate (55.4% versus 58.7%) when fresh or vitrified donor oocytes were

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compared [71].

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Future perspectives

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Considering the high oocyte survival rates and subsequent developmental competence

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reported by leading clinics in the field, radical improvement in outcome cannot be expected

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during the subsequent decade. Advancement should be achieved in the widespread

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application in all clinics and for all cases where oocyte vitrification will be beneficial, and

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efforts should be made to increase the average results to the level that is now the privilege of

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a limited number of laboratories. Standardisation, selection of the best method, and thorough

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education of all operators involved may result in considerable increases in efficiency.

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However, due to the lack of consensus between scientists, suppliers and clinics, as well as the

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slowly emerging educational network, this process is slow.

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The intrinsic handicaps of current vitrification techniques, in which the primitive

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manufacturing process relies on the manual skills of the embryologists, should be eliminated.

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Efficiency, consistency, reliability and safety - for operators, patients, oocytes and embryos -

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can only be guaranteed by changing our approach. Automation of vitrification is an absolute

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necessity.

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The priority should be the automation of equilibration and cooling, and focus on warming

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and dilution in a subsequent phase. Optimally, an automated vitrification machine should

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meet the following requirements.

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1. High throughput - to decrease the total working time per vitrified sample by 50%

397

(preferably 75% or more)

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2. Disposable devices to hold and move samples during equilibration, loading and cooling

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3. The use of variable volumes of vitrification/warming solutions (according to the

400

requirement of the laboratory)

401

4. Variability in the equilibration protocol (one-two-three phase; stepwise or continuous

402

increase in concentration)

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5. Visual microscopic control of all oocytes or embryos during the whole equilibration

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process recorded for documentation

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6. Preferably bulk equilibration of all embryos / oocytes of an individual patient, then

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selection / pairing before loading - this arrangement would spare time and help to select

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samples immediately before cooling

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ACCEPTED MANUSCRIPT 7. High-rate aseptic cooling - with minimal liquid nitrogen consumption - in the same device

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that is used for equilibration or a separate carrier tool loaded automatically

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8. Safe aseptic storage option that (preferably) accommodates to the currently available liquid

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nitrogen containers

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9. Automated transfer of samples from the vitrification machine to storage containers

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10. Easy and safe handling

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11. Preferably an automatic oocyte / embryo labeling-tracking system [33,66] that may help

415

to avoid any mixing between patients and between oocytes and embryos of the same patient,

416

especially after PGD/PGS

417

During the past 10 years, several publications have described solutions that may meet some

418

of the criteria, but a complex solution is still missing. The only commercially available

419

vitrification machine [81] may be a step forward, but does not meet several requirements (for

420

example visual control, selection after equilibration).

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The involvement of microfluidics for equilibration may offer many benefits, but may make

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the equipment complicated and difficult to use. Future approaches may require more

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creativity and simple solutions for the seemingly complicated problems that slow down

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advancement. The rapid advancement of nanotechnology and microdevices, and the

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determined efforts of some innovative embryologists may offer a feasible prototype in the

426

very near future.

427

Conclusion: where efficiency and safety meet

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The optimal oocyte vitrification technique should be a balance between efficiency and safety.

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The ultimate goal would be to achieve as many healthy live births as possible with the lowest

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amount of oocytes. While equal survival rates between open and closed devices may be

431

obtained, it seems that the oocytes in the closed devices are more susceptible to

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ultrastructural changes and could have a slightly impaired development. The reduced

433

blastocyst formation with oocytes from infertile patients may be attributed to an inherent

434

lower oocyte quality in an infertile population that suffers even more after the vitrification

435

procedure. It is still unclear whether these changes are clinically relevant or not since equal

436

clinical pregnancy rates seem to be obtained. In general, it is accepted that very high cooling

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and warming rates (i) produce less cryodamage to the oocyte, (ii) have less negative impact

438

on embryo development and (iii) generate higher delivery rates.

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ACCEPTED MANUSCRIPT The only devices that guarantee a contamination-free procedure are the closed devices.

440

However, since no report has ever been made on cross-contamination with reproductive cells,

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the question remains whether it is truly needed to close the devices before any contact with

442

LN2. Instead of introducing further versions of existing techniques, research should rather

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focus on automation of the whole equilibration and vitrification procedure that combines

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sterility with a high cooling rate.

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The obstetric and neonatal outcomes appear to be similar when fresh or vitrified oocytes are

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compared (heterologous and autologous oocytes). In the case of heterologous oocytes,

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adverse outcomes are expected due to immunological foreign fetus, which is not the case

448

when autologous oocytes are used.

449

It also seems important that a donor has a known fertility before she starts donating. If a

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young donor, after multiple stimulations, appears to be infertile herself, she might be at

451

increased risk of developing breast or gynaecological cancers. For the operator, a drastic

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reduction of the dangers can only be expected when automated vitrification machines are

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introduced.

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It is clear that more research is required (i) to find the optimal balance in which the efficiency

455

of vitrification is not affected by the safety and (ii) to develop new technical solutions.

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Acknowledgements

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We would like to thank the reviewers for their nice and thorough review.

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References

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Investigated parameters Method Device Effect Reference spindle Polscope Cryoloop spindle is maintained at 37°C after vitrification [51] spindle recovery Polscope HSV vit and warming at 37°C: fastest recovery [18] spindle Polscope Cryotip 88% spindle positive oocytes [67] spindle Polscope HSV high spindle re-appearance post warming [26] spindle Polscope Cryotop repolimerized within 3 hours [65] spindle/metaphase plate CM Cryotip fresh ≈ vitrified (3h culture) [19] spindle; chromosme alignment CM Cryoleaf fresh = vitrified [11] spindle CM Cryoleaf bipolar organization maintained; chromosome alignment compromised [24] spindle TEM Cryotip spindle alterations reversed upon warming [6] vacuoles TEM Cryoleaf and Cryoloop fresh ≈ vitrified [64] vacuoles TEM Cryotip fresh < vitrified [38] vacuoles TEM Cryotip slightly higher vacuolization (compared to fresh) [6] vacuoles TEM fresh < Cryotop [68] Cryotop mitochondria TEM Cryoleaf and Cryoloop fresh = vitrified [64] mitochondria TEM Cryotip fresh > vitrified (degeneration) [38] mitochondria TEM Cryotip fresh = vitrified [6] M-SER TEM Cryoleaf and Cryoloop fresh > vitrified (smaller size and slender shape) [64] M-SER TEM smaller [68] Cryotop M-V TEM fresh = vitrified [68] Cryotop M-SER and M-V TEM Cryotip fresh = vitrified [6] mitochondria staining Cryotop fresh = vitrified [65] ATP production BA Cryoleaf ATP production ↓; ATP synthesis recovers a;er 180h [53] redox homeostasis CM Cryotop vitrified oocytes: oxidized state [65] mitochondrial membrane potential CM Cryoloop mitochondrial membrane potental restored 4 hours after vitrification [15] cortical granules TEM Cryoleaf and Cryoloop fresh > Cryoleaf > Cryoloop [64] cortical granules TEM Cryotip fresh > vitrified [38] cortical granules TEM Cryotip amount and density: fresh > vitrified [6] cortical granules TEM discontinued stratification [68] Cryotop microvilli TEM Cryoleaf and Cryoloop fresh ≥ vitrified [64] microvilli TEM 30% abnormal [68] Cryotop intracellular Ca response FM Cryotip fresh ≈ vitrified [38] intracellular Ca response FM HSV altered Ca oscillations (longer period, higher amplitude, lower frequency) [63] ultrastructure TEM Cryotop and Cryotip rehydration: open > closed; ultrastructural preservation: open > closed [8] 8 genes RT-PCR HSV fresh = vitrified [30] mRNA (18 genes) RT-PCR Cryotop vitrification maintains 63.3% of the mRNA content [12] gene expression microarray Cryotip downregulation of specific transcripts (loss/alteration of mRNA content) [61] DNA fragmentation TUNEL fresh = vitrified Cryotop [55] second meiosis arrayCGH HSV fresh = vitrified [27] HSV: High Security Vitrification; M-SER: mitochondria-smooth endoplasmic reticulum complex; M-V: mitochondria-vacuole complex; TEM: tunnel electron microscopy; CM: confocal microscopy; RT-PCR: reverse transcriptase-polymerase chain reaction; FM: (epi)fluorescence microscopy; BA: bioluminescent assay; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling ; * unfertilized mature oocytes; ** in vitro matured oocytes; *** in vitro and in vivo matured oocytes.

ACCEPTED MANUSCRIPT Investigated parameters

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Oocyte source Effect

Reference

Development Development Development Development Development Development Development Development Development Development Ploidy Ploidy Metabolic profile DNA (hydroxy)methylation

fixed time point fixed time point fixed time point fixed time point fixed time point fixed time point fixed time point fixed time point Time lapse Time lapse FISH micrarray based CCS LC-MS CM

Cryotop HSV Vitrisafe (open versus closed) HSV versus Cryotop Cryotop Cryotop Cryotop Vitri-Ingá Cryotop HSV Cryotop Cryotop Cryotop HSV

donor donor donor donor infertile infertile infertile infertile infertile donor infertile infertile donor donor

[19] [26] [70] [29] [60] [74] [77] [2] [13] [27] [48] [34] [32] [27]

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fresh = vitrified fresh = vitrified open = closed Cryotop: higher blastomere number fresh = vitrified fresh = vitrified fresh = vitrified fresh: higher blastomere number vitrification: faster syngamy (3h) fresh = vitrified 5/5 euploid fresh = vitrified alpha-CEHC: higher in embryos from vitrified oocytes fresh = vitrified

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FISH: fluorescent in situ hybridisation; CCS: comprehensive chromosome screening; LC-MS: liquid chromsatography coupeld with mass spectrometry; alpha-CEHC: 2,5,7,8-tetramethyl-2-(2'carboxyethyl)-6-hydroxychroman; CM: confocal microscopy; HSV: high security vitrification