European Journal of Obstetrics & Gynecology and Reproductive Biology
Full length articleVitrification and conventional freezing methods in sperm cryopreservation: A systematic review and meta-analysis
Introduction
As an efficient method, sperm cryopreservation is an essential aspect of assisted reproductive technique (ART) and male fertility preservation. The number of patients with azoospermia and oligospermia avoid repeated biopsies or aspirations through this technique [1]. Cancer patients also preserve their fertility by this technique before undergoing chemotherapy or radiation. There are several methods which differ from each other in terms of freezing rates, composition of cryoprotectants (CPAs), dilution rates, carrier tools and thawing protocol. Two principal approaches for sperm cryopreservation have been adopted: conventional freezing and vitrification.
There are two conventional freezing methods used in sperm cryopreservation: slow freezing and vapor fast freezing. Slow-freezing consists of 2∼3 steps and usually costs 2∼4 h to complete [2]. Firstly, spermatozoa accumulating by ejaculation or other techniques keep at room temperature for 10 min. Secondly, samples are gradually frozen from 20 °C to 5 °C at a rate of 0.5–1 °C/min and then cooled from 5 °C to −80 °C at rate of 1–10 °C/min. Finally, samples are plunged into liquid nitrogen [3]. Vapor fast freezing is based on a direct contacting of samples to liquid nitrogen vapors for 10–15 minutes [4]. Conventional freezing methods involve the use of CPAs to minimize osmotic damage and to avoid intracellular and extracellular ice crystal formation, but can be cytotoxic [5]. There are two categories of CPAs, one is permeable CPAs including dimethyl sulfoxide, glycerol, glycol, ethylene and methanol, the other is nonpermeable CPAs including albumins, dextrans and egg yolk citrate [6]. It is well known that permeable CPAs stabilize the plasma membrane and nonpermeable CPAs minimize the intracellular ice formation. However, in most cases, nonpermeable play a supporting role that augmented the effectiveness of permeable CPAs [7].
Vitrification is a process to solidify liquid into an amorphous or glassy state [8]. This approach was first proposed in 1937, but at that time, the cooling rate could not meet the vitrification requirement [9]. Currently, vitrification is the most commonly used technique in the preservation of oocytes and embryos, which required addition of high concentration of permeable CPAs [10]. However, this technique cannot successfully preserve mammalian spermatozoa because spermatozoa have higher osmotic fragility than other reproductive tissues [11]. Until 2002, a new vitrification protocol named cryoprotectant-free vitrification (CPA-free) was introduced by Nawroth et al, which differed from former vitrification protocols in absence of high concentrations of permeable CPAs [12]. In the last 16 years, several types of pre-cooling techniques, carrier tools, thawing methods have been introduced to improve the efficacy of vitrification protocol [13]. However, studies that evaluated the efficacy of sperm vitrification showed rather conflicting results.
To the best of our knowledge, this is the first meta-analysis to compare the safety and efficacy of two different freezing techniques used in sperm cryopreservation. The objective of this study was to perform a meta-analysis of the randomized controlled trials that evaluated the efficacy of sperm vitrification in terms of total motility, progressive motility, morphology and DFI compared with conventional freezing methods.
Section snippets
Types of studies
The inclusion criteria of the selected studies were defined a priori during the design phase of this systematic review. Due to the inherent methodological limitations and low overall quality of observational studies, only randomized controlled trials (RCTs) using human spermatozoa and reporting the sperm quality parameters, including total motility, progressive motility, morphology, or DNA fragmentation index (DFI), were selected. Studies published in English language were selected. No date or
Statistics analysis
Weighted mean differences (MD), which were identified with 95% confidence intervals (95% CI), were used to analysis the all included sperm parameters in the different comparison groups. Heterogeneity was assessed through I2 statistic (an I2 of<50%, 50.0–75.0% and >75.0% indicating low, moderate and high heterogeneity, respectively) [15]. A fixed-effects model was used when I2<50% and random-effects model was used when I2>50.0%. We conducted all data analyses through STATA software 12 (Stata
Results
The research strategy yielded a total of 2428 articles, of which 27 studies were initially identified for full reading as they did fulfill the selection criteria (Fig. 1). Of the remaining 27 studies, 14 studies were excluded after reading the full manuscripts. Reasons for not including the other studies were: (i) insufficient date for analysis (n = 3) [[17], [18], [19]]; (ii) no conventional freezing group (n = 5) [[20], [21], [22], [23], [24]]; (iii) no vitrification group (n = 6) [[25], [26]
Discussion
Our meta-analysis is the first attempt to evaluate and compare the efficacy with two different cryopreservation methods used for human spermatozoa. The principal results of this meta-analysis suggests that spermatozoa coming from vitrification cycles could result in better total and progressive motility than those coming from conventional freezing cycles. This study also reveals that vitrification protocol and cryopreservation of different quality sperm at vitrification/thawing cycles influence
Funding
This research was financially supported by National Natural Science Funding of China (No. 81673224; 81273018; 30700654), Natural Science Funding of Shaanxi Province (No.2015JM8436), Science Funding of Health Department, Shaanxi Province (2012D58) and the Fundamental Research Funds for the Central University (XJJ 2011024) and the Project of Independent Innovative Experiment for Postgraduates in medicine in Xi’an Jiaotong University (Grant No. YJSCX-2018-011).
Conflict of interest
The authors declare that no competing interests exist.
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