Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

Table 2

(a)–(c) Evaluation of DNA integrity after cryopreservation: description of the experimental design and conclusions.

(a)

Authors	Test to evaluate DNA integrity	Number of samples	Cryopreservation method	“Does the freezing-thawing procedure induce sperm DNA damage?”
Hamamah et al. [94]	Acridine orange staining and Feulgen-DNA quantitative microspectrophotometry	10	Unspecified	Yes
Spanò et al. [44]	SCSA + Acridine orange staining	19	Equilibration at 37°C, freezing in liquid nitrogen vapour at −80°C and then storage in liquid nitrogen at –196°C	Yes
Hammadeh et al. [95]	Acridine orange staining	59	Computerized slow-stage freezer + static liquid nitrogen vapour	Yes
Donnelly et al. [6]	COMET assay	40	Equilibration at 37°C, freezing in liquid nitrogen vapour at −80°C and then storage in liquid nitrogen at –196°C	Yes
Gandini et al. [96]	Acridine orange staining	19	Equilibration at 37°C, freezing in liquid nitrogen vapour at −80°C and then storage in liquid nitrogen at –196°C	Yes
de Paula et al. [40]	TUNEL assay	77: (i) 30 normozoospermic (ii) 47 oligozoospermic	Use of freezer at –20°C, freezing in liquid nitrogen vapour, then storage in liquid nitrogen –196°C	Yes
Petyim and Choavaratana [43]	Acridine orange staining	50	Freezing with liquid nitrogen vapour + computerized program freezer	Yes
Nagamwuttiwong and Kunathikom [97]	Acridine orange staining	20	Freezing with liquid nitrogen vapour	Yes
Dejarkom and Kunathikom [98]	Acridine orange staining	20	Computerized controlled rate freezing	Yes
Thomson et al. [46]	TUNEL assay	60	Use of programmable freezer	Yes
Thomson et al. [46]	TUNEL assay	320	Sample frozen with and without cryoprotectant by slow-controlled-rate method using a programmable freezer	Yes
Zribi et al. [45]	TUNEL assay	15	Equilibration at 37°C, freezing in liquid nitrogen vapour at −80°C, then storage in liquid nitrogen at –196°C	Yes

(b)

Authors	Test to evaluate DNA integrity	Number of samples	Cryopreservation technique	“Does the freezing-thawing procedure induce sperm DNA damage?”
Donnelly et al. [6]	COMET assay	57: (i) 17 fertile (ii) 40 infertile	Equilibration at 37°C, freezing in liquid nitrogen vapour at −80°C, then storage in liquid nitrogen at –196°C	Yes, but semen from fertile men appears to be more resistant to freezing damage
Kalthur et al. [49]	COMET assay + Acridine orange staining	44	Equilibration at 37°C, static cooling at 4°C, cooling vapour phase, then storage in liquid nitrogen at –196°C	Yes, but morphologically abnormal sperms seems to be less resistant to freezing damage
Ahmad et al. [99]	COMET assay	196: (i) 30 normospermic (ii) 166 infertile	Freezing with static-phase vapour cooling procedure	Yes, but the sperm DNA integrity of frozen samples of fertile men is higher

(c)

Authors	Test to evaluate the DNA integrity	Number of samples	Cryopreservation technique	“Does the freezing-thawing procedure induce sperm DNA damage?”
Høst et al. [100]	Immunoperoxidase detection of digoxigenin-labelled genomic DNA	53: (i) 20 fertile (ii) 33 infertile	Conventional cryopreservation	No
Steele et al. [101]	COMET assay	21: (i) 9 control (ii) 12 with obstructive azoospermia	Freezing in liquid nitrogen vapour	No
Duru et al. [41]	TUNEL assay + annexin V	21	Equilibration at 37°C, freezing in liquid nitrogen vapour at −80°C, then storage in liquid nitrogen at –196°C	No
Isachenko et al. [42]	COMET assay	18	Programmable slow freezing + vitrification	No
Paasch et al. [50]	TUNEL assay + flow cytometric kit for apoptosis	84	Freezing at –20°C, freezing in liquid nitrogen vapor at –100°C, then storage in liquid nitrogen at –196°C	No