Sperm vacuoles are not modified by freezing thawing procedures

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1 Reproductive BioMedicine Online (2013) 26, ARTICLE Sperm vacuoles are not modified by freezing thawing procedures Nicolas Gatimel, Roger Leandri *, Jean Parinaud Centre d Assistance Médicale à la Procréation, Hôpital Paule de Viguier, 330 avenue de Grande Bretagne, Toulouse, France; Université de Toulouse; UPS; Groupe de Recherche en Fertilité Humaine (EA 3694, Human Fertility Research Group), 330 avenue de Grande Bretagne, Toulouse, France * Corresponding author. address: leandri.r@chu-toulouse.fr (R Leandri). Dr Nicolas Gatimel obtained a specialist degree in medical biology at Toulouse University in His 4-year residency in medical biology specialized in reproductive biology, especially assisted reproductive technologies, was supervised by Jean Parinaud at Paule de Viguier Hospital. He has also obtained a MSc at Paris VII University. Abstract Since the development of the motile sperm organellar morphology examination (MSOME) in 2001 for observing the cephalic vacuoles at high magnification, no study as yet assessed the effect of cryopreservation on these vacuoles, although sperm freezing thawing procedures are known to affect sperm quality. Examination of the vacuoles before and after freezing thawing would indicate whether the same normality criteria can be applied for frozen as for fresh spermatozoa when performing intracytoplasmic morphologically selected sperm injection. In 27 sperm samples from fertile men, analysis of conventional sperm parameters (motility, vitality, percentage of normal forms) and a morphological analysis at high magnification ( 6000) using image analysis software was performed before freezing and after thawing. Whereas there were expected decreases in motility (P < ), vitality (P < 0.001) and percentage of normal forms (P < 0.05) after cryopreservation, there was no evidence for any difference in any vacuolar criteria (relative vacuole area, total vacuole area, vacuole area in the anterior, median and basal parts of the head, percentage of spermatozoa with a vacuole area 6.5% and percentage of spermatozoa with a vacuole area >13%). Freezing thawing procedures have no effect on human sperm vacuoles. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: freezing, IMSI, MSOME, spermatozoa, thawing, vacuoles Introduction Nomarski interference contrast microscopy at high magnification ( 6000 to 10,000) revealed a new morphological criterion in human spermatozoa: the presence of nuclear vacuoles. Real-time observation of motile spermatozoa by this technique, motile sperm organellar morphology examination (MSOME), was first used by Bartoov et al. (2001) to select spermatozoa with as few vacuoles as possible before microinjection into the oocyte by intracytoplasmic morphologically selected sperm injection (IMSI). IMSI may improve the pregnancy rate compared with conventional ICSI, mainly /$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

2 Same criteria can be used for IMSI with fresh and thawed spermatozoa 241 after several failures of ICSI, as confirmed by a prospective randomized controlled trial (Antinori et al., 2008). The origin of these vacuoles raises many questions. Their frequency and their great heterogeneity in area and location may indicate that they do not all have the same origin and the same pathological significance. Most studies that have attempted to determine the origin of these vacuoles argue in favour of a nuclear origin. Several studies have found increased levels of fragmented DNA in spermatozoa with large vacuoles (Boughali, 2006; Franco et al., 2008; Garolla et al., 2008; Oliveira et al., 2010) whereas others (Boitrelle et al., 2011; Perdrix et al., 2011) show abnormalities of chromatin condensation in such spermatozoa. Other authors, however, consider that the vacuoles may be of acrosomal origin (Kacem et al., 2010), although it was recently shown that acrosome-less spermatozoa from globozoospermic patients present vacuoles similar to those from fertile men (Gatimel et al., 2013). Cryopreservation of human spermatozoa is a technique that has been routinely used for many years (Sherman, 1954), in particular to preserve fertility before treatment that may be gonadotoxic or for sperm donation. Despite the success of cryopreservation of spermatozoa in assisted reproductive technology, the freezing thawing process is undoubtedly associated with alterations of sperm quality. The impairment the most often described in the literature is decreased motility (Pilikian et al., 1982; Serafini et al., 1986; Smith and Steinberger, 1973). The main morphological abnormalities reported after freezing thawing are coiled tails (Critser et al., 1988), acrosomal defects (Cross and Hanks, 1991; Royere et al., 1996) and damage to membrane structures (Check et al., 1991; Royere et al., 1996). The primary cause of cell injury during cryopreservation is the formation of intracellular ice crystals (Muldrew and McGann, 1990; Watson, 1995). In addition, during cooling, crystallization of the extracellular medium increases the concentration of solutes in suspension in the liquid phase and therefore the osmolarity of the extracellular medium, which may lead to excessive intracellular dehydration (Donnelly et al., 2001). These different physical events are associated with mechanical damage during cold shock and so may result in rupture of the plasma membrane and disturbance of cellular organelles. The aim of this work was to evaluate the impact of freezing thawing on the sperm head vacuoles in association with conventional sperm parameters in order to discover whether the same normality criteria can be used for frozen spermatozoa (in particular in IMSI) as for fresh spermatozoa. Materials and methods Study design The subjects were selected between September 2011 and April The population studied consisted in 27 men aged from 23 to 49 years (mean ± SD age 33.7 ± 6.0 years) with proven spontaneous fertility (time to conception 4.3 ± 5.1 months). All subjects gave their informed consent to participate in the study. The protocol was approved by the Committee for the Protection of Persons Participating in Biomedical Research Programmes (reference no /Avis no. 1, approved 8 July 2011). Semen samples were collected by masturbation after 2 5 days of sexual abstinence and were processed for analysis after liquefaction for 20 min at 37 C. The unselected fresh spermatozoa were subjected to a sperm count, motility, vitality and morphology analysis as well as a detailed morphometric analysis of the vacuoles at high magnification using image analysis software. An aliquot of fresh spermatozoa was cryopreserved for at least 1 month and then thawed. After thawing, the following analyses were carried out: motility, vitality, morphology and high-magnification examination of vacuoles. The significant variations observed after freezing thawing in motility, vitality and percentage of normal forms led to the study of these parameters for six samples in a secund treatment: after exposure to cryoprotectant without freezing. An aliquot of fresh spermatozoa was exposed to cryoprotectant for 10 min, a period equivalent to that required for the freezing process; then the analyses were carried out after removal of the cryoprotectant as for thawing. Conventional sperm parameters Semen evaluation was performed according to the standard methodology proposed by the World Health Organization guidelines (WHO, 2010). Furthermore, sperm morphology was explored according to Kruger s classification (Kruger et al., 1995). For the sperm morphology, the smears were air dried for 10 min then stained using the Diff-Quick procedure (Dade, Düdingen, Switzerland). A Hamilton Thorn version 12.3 HTM-IVOS analyser was used. Each spermatozoon was identified and then analysed by computerized software according to strict criteria (Kruger et al., 1995; Marnet et al., 2000). Freezing The cryoprotectant SpermFreeze (FertiPro, Beernem, Belgium) containing 15% glycerol was added dropwise to the volume of fresh spermatozoa to be frozen at a ratio of 0.7 ml cryoprotectant/ml spermatozoa. The diluted spermatozoa were then placed in high-security CBS straws, labelled and sealed by an automated MAPI system (CryoBioSystem, St-Ouen-sur-Iton, France). Cooling was carried out in a programmable freezer (Minicool 40 PC; AirLiquide, Marnela-Vallée, France). The programme was as follows: initial temperature +20 C, then 5 C/min down to 8 C, seeding at 8 C, 10 C/min to 30 C and 25 C/min to 150 C. At completion of the freezing program, the sperm straws were immersed and stored in liquid nitrogen. Thawing The sperm straw was left at room temperature for 2 min. Then the contents were emptied into a 2.5 ml plastic tube, and 2 ml G-IVF washing solution (Vitrolife, Göteborg, Sweden) was added drop by drop. Washing was carried out by centrifugation for 5 min at 400g. The pellet was resuspended in 200 ll G-IVF. Motility and vitality were then assessed and followed by a standard sperm morphology analysis and examination at 6000 magnification by interference contrast microscopy.

3 242 N Gatimel et al. High-magnification morphological examination Fresh or thawed spermatozoa (50 ll) were washed in 2.5 ml G-IVF washing solution by centrifugation for 5 min at 400g. The pellet was resuspended in 100 ll G-IVF and the spermatozoa were fixed by addition of 100 ll of 3.7% PBS-formaldehyde. An aliquot (2 ll) of this suspension was placed in a glass-bottomed dish (WillCo-dish; WillCo Wells BV, The Netherlands) and examined by Nomarski interference contrast microscopy with a Leica DFC-280 camera mounted on a Leica DMI 6000 microscope with an immersion objective lens 100 and camera magnification 1. For each sample, sperm head vacuoles were analysed on 100 spermatozoa that were randomly photographed and separately analysed using digital imaging system software (Leica Application Suite Interactive Measurement version 3.6; Leica). Measurements using the software were carried out by a single operator who was blinded to the origin (fresh or thawed) of the sample. The Leica LAS Interactive Measurement module allows measurement of sperm head areas and vacuole areas by manually depicting their outline. The area and position of each vacuole were recorded (Figure 1). Relative vacuole area is the ratio of the area of all the vacuoles of a spermatozoon to the area of its head. Statistical analysis Data are means ± SD. Statistical analysis was performed with Statview for Windows (Abacus Concepts, Berkeley, CA, USA). Student s paired t-test or Wilcoxon s paired t-test were used for comparison of all parameters before freezing, after cryoprotectant and after freezing thawing depending on the normality of distribution of the variables. Results Conventional sperm parameters Mean sperm concentration was 87 ± 56 million/ml. Progressive motility decreased significantly after freezing thawing (49 ± 15% versus 25 ± 12%; P < ; Figure 2A) and also after exposure to cryoprotectant alone (47 ± 17% versus 36 ± 21%; P < 0.05; Figure 2B). Freezing was associated with decreased motility compared with cryoprotectant exposure alone (36 ± 21% versus 16 ± 11%; P < 0.05; Figure 2B). Vitality decreased significantly after freezing thawing (77 ± 9% versus 46 ± 9%; P < 0.001; Figure 2A); but also after exposure to cryoprotectant alone (78 ± 8% versus 71 ± 14%; P < 0.05; Figure 2B). Freezing was associated with decreased vitality compared with exposure to cryoprotectant alone (71 ± 14% versus 51 ± 11%; P < 0.05; Figure 2B). The percentage of normal forms decreased significantly after freezing thawing (17.8 ± 6.5% versus 14.9 ± 7%; P < 0.05; Figure 2A). The abnormalities that were significantly increased after freezing thawing were (percentage of spermatozoa with bent tails (4.1 ± 2.7% versus 6.2 ± 2.8%; P < 0.01), coiled tails (0.5 ± 0.8% versus 3.4 ± 2.3%; P < ) and round shape (4.8 ± 4.7% versus 7.5 ± 6.2%; P < 0.01). There was no significant variation of the percentage of normal forms in the second series of experiments (Figure 2B) with exposure to cryoprotectant alone. Vacuoles observed at high magnification by Nomarski interference contrast microscopy The relative vacuole area remained unchanged after freezing thawing (6.2 ± 1.8% versus 6.1 ± 1.7%; not significant; Table 1). Similarly, the total absolute vacuole area and anterior, median and posterior areas were unchanged after freezing thawing. The percentage of spermatozoa with small vacuoles or no vacuoles (relative vacuole area 6.5%) remained unchanged after freezing (63.5 ± 16.1% versus 63.4 ± 13.5%). Similarly, the percentage of spermatozoa with large vacuoles was not changed by freezing (9.2 ± 7.2% versus 8.8 ± 6.8%). Discussion As far as is known, 10 years after the development of a new concept called MSOME for observing the sperm head Figure 1 Example of measurement of the areas of the sperm head vacuoles with Leica Application Suite version 3.6. The head of this spermatozoon measures 13.8 lm 2 ; it has an anterior vacuole of 0.28 lm 2 and a median vacuole of 0.58 lm 2. To indicate the location of the vacuoles, the sperm head is divided into three equal parts along its length and the position of the vacuole is given by its centre.

4 Same criteria can be used for IMSI with fresh and thawed spermatozoa 243 Progressive motility (%) A P < B P < 0.05 P < 0.05 P < 0.05 Vitality (%) A P < B P < 0.05 P < 0.05 P < 0.05 A Normal forms (%) B NS P < 0.05 NS NS Before freezing After cryoprotectant After freezing / thawing Figure 2 Progressive motility, vitality and percentage of normal forms before and after freezing in 27 individuals (A) and before freezing, after cryoprotectant and after freezing in six individuals (B). Table 1 Vacuolar parameters before and after freezing thawing in 27 fertile sperm samples. Before freezing After freezing thawing Vacuole area Total (lm 2 ) 0.75 ± ± 0.20 Anterior (lm 2 ) 0.24 ± ± 0.13 Median (lm 2 ) 0.48 ± ± 0.14 Basal (lm 2 ) 0.04 ± ± 0.03 Relative vacuole area (%) 6.2 ± ± 1.7 Spermatozoa according to relative vacuole area (%) 6.5% 63.5 ± ± % 27.3 ± ± 8.3 >13% 9.2 ± ± 6.8 Values are mean ± SD. There were no statistically significant differences between the two groups.

5 244 N Gatimel et al. vacuoles by high magnification with Nomarski contrast before IMSI (Bartoov et al., 2002), there is no data on the use of this detailed morphological analysis after sperm cryopreservation in liquid nitrogen. Regarding conventional sperm parameters, this study observed a significant decrease of 49% in progressive motility after freezing thawing, as already well described in the literature (Cohen et al., 1981; Hammadeh et al., 2001; Keel et al., 1987; Mossad et al., 1994; Verheyen et al., 1993). The decrease in motility is of course partly due to a loss of vitality: a 40% decrease in vitality was observed after freezing thawing (Figure 2). However, the fact that the motility/vitality ratio decreased significantly after freezing thawing (0.64 ± 0.18 versus 0.51 ± 0.22; P < 0.05) reflects a specific effect of the freezing process on motility which is additional to its effect on vitality. Closer analysis of the stages of the freezing procedure in a subpopulation of six fertile men revealed a specific effect of exposure to cryoprotectant and its removal on the decreases in vitality and motility, an effect which was however less than that of freezing itself (Figure 2). The use of a cryoprotectant limits cell damage. However, the addition and removal of the cryoprotectant creates osmotic stress. The cellular changes related to this osmotic stress may be due to: (i) some damage caused by the interaction between the components of the cytoskeleton that resist cell shrinkage and the plasma membrane itself (Gao et al., 1995) in hyper-osmotic conditions (when cryoprotectant is added); and (ii) membrane rupture in hypo-osmotic conditions when the cryoprotectant is diluted during thawing (friction forces at the membrane pores created by the flow of water and cryoprotectant). In addition, the same team demonstrated that motility is more sensitive than membrane integrity to variations in osmolarity. This suggests that cellular structures involved in motility other than plasma membrane are directly affected by these variations in cell volume related to the cryoprotectant; one hypothesis is mitochondrial impairment (Gao et al., 1995). The percentage of normal forms was significantly lower after freezing, which is also in agreement with the data of the literature. After freezing thawing, the percentage of normal forms is decreased by accepted 15% to 62.5%, depending on the authors (Donnelly et al., 2001; Hammadeh et al., 1999, 2001; Verheyen et al., 1993). Authors who have examined the ultrastructure of the spermatozoa report plasma membrane swelling, acrosomal swelling or loss and pulling back of the mitochondrial sheath (Royere et al., 1996). Acrosomal changes and swelling of the subacrosomal space due to detachment between the internal acrosomal membrane and the nuclear membrane have also been described by electron microscopy after freezing thawing (Ozkavukcu et al., 2008). To study the impact of cryopreservation on vacuoles, it seemed indispensable to use an objective tool which enables precise calculation of the areas of the vacuoles by depicting their outline. The drawback of this method is that it is extremely time consuming. It takes at least 30 min to photograph 100 spermatozoa per individual and 90 min for morphometric analysis of these 100 spermatozoa using the software. On the other hand, not using such a precise image analysis tool but only the naked eye is probably responsible for the existence in the literature of many varying definitions of the large vacuoles, which appear to be the most harmful: for authors that use image analysis software, these are vacuoles that occupy more than 13% of the area of the sperm head (Perdrix et al., 2011); for those that do not, the threshold can range from 25% (Boitrelle et al., 2011) to 50% (Franco et al., 2008; Mauri et al., 2010; Oliveira et al., 2010). The present study chose to use the relative vacuole areas with the thresholds used by Perdrix et al. (2011) because it is the only team that defined their thresholds based on different clinical phenotypes and employing image analysis software. According to these authors, the relative vacuole area is the most discriminant criterion between normal and abnormal spermatozoa (Perdrix et al., 2012). Finally, the present study found no difference in the vacuole criteria before and after freezing thawing in terms of relative vacuole area, total vacuole area or vacuole area in the anterior, median and basal parts of the head, nor in terms of distribution of the subpopulations of spermatozoa with small or large vacuoles. The freezing process (dehydration and rehydration of the cell, formation of intracellular crystals and cooling shock) thus has no impact on the sperm cephalic vacuoles. A limitation of this study is that it involved only fertile men. Indeed, a higher susceptibility to freezing was found in infertile men when compared with fertile men for criteria such as chromatin condensation (Hammadeh et al., 1999). Furthermore, DNA integrity is more affected by freezing in teratospermic when compared with normospermic men (Kalthur et al., 2008). It could therefore be postulated that the impact of freezing and thawing on fine sperm head morphology could be less detectable in this population of fertile men. Further studies are therefore needed on an infertile population. However, it must be highlighted that, although less susceptible, all sperm parameters affected by the freeze thawing procedure in infertile men are also affected in fertile men (Hammadeh et al., 1999, 2001; Verheyen et al., 1993), while the vacuolar criteria are remarkably stable. These results are in disagreement with those of a recent publication showing that cryopreservation induces sperm nuclear vacuolization and decondensation (Boitrelle et al., 2012). The main difference between the two studies is that Boitrelle et al. studied men from infertile couples, although their sperm count and motility were in the normal range, while only samples from recently fertile men were included in the present study. Moreover, some technical differences between the studies could explain the opposite conclusions: (i) the dilution ratio with the cryoprotectant was different; and (ii) Boitrelle et al. used a morphological classification (Vanderzwalmen et al., 2008) that includes not only vacuoles but also other sperm abnormalities. In conclusion, this study demonstrates that the cryopreservation procedures have no effect on human sperm vacuoles and argues for the use of the same vacuole criteria as those established from fresh semen for IMSI with frozen thawed spermatozoa. Acknowledgements The authors thank all of the volunteers who participated in this study. Mrs Françoise Cendres is also acknowledged for her assistance.

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7 246 N Gatimel et al. number of nuclear vacuoles. Reprod. Biomed. Online 17, Verheyen, G., Pletincx, I., Van Steirteghem, A., Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm. Hum. Reprod. 8, Watson, P.F., Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7, WHO, WHO Laboratory Manual for the Examination of Human Semen and Sperm-cervical Mucus and Interaction. World Health Organization, Cambridge. Declaration: The authors report no financial or commercial conflicts of interest. Received 26 August 2012; refereed 23 November 2012; accepted 27 November 2012.