Nothing Special   »   [go: up one dir, main page]
Skip to main content
Middle East Fertility Society Journal
Download PDF

Comparison between open and closed systems for vitrification of individual sperm: assessing morphometric measurements and chromatin integrity

Middle East Fertility Society Journal volume 29, Article number: 39 (2024) Cite this article

Abstract

Background

Classic vitrification methods are not appropriate when there are minimal numbers of viable sperm, and the new methods emphasize the low semen volumes in these cases. The aim was to assess the efficacy of the cryotech as a device for freezing low sperm volume, through the two methods of open (OVS) and closed (CVS) vitrification systems.

Methods

Testicular biopsy samples from 30 men with obstructive azoospermia (OA) were assigned to three groups fresh control (FC), OVS, and CVS. Testicular sperms were selected using an ICSI injection pipette and vitrified on the cryotech straws, containing one droplet of freezing medium. After warming, sperm head morphometric characterizations were evaluated with the MSOME technique. Sperm motility, membrane integrity, chromatin quality assessment including DNA fragmentation, Chromomycine A3 staining (CMA3), and Aniline Blue (AB) were assessed. Fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) was done to examine sperm acrosome integrity.

Results

The mean sperm motility, viability, and sperm with intact acrosome reduced after vitrification, in both methods of CVS, and OVS, but the results were more promising in the closed method (p < 0.05). However, the variations were not significant between the two methods of cryopreservation, the OVS undergoes significant head dimensions changes compared to the control group (p < 0.05). The results also showed higher membrane, and chromatin abnormality after OVS (p < 0.05).

Conclusions

The overall post-thaw recovery of human testicular sperm proposes that CVS is more efficient for single sperm cryopreservation, while higher sperm viability, and lower alterations in chromatin, acrosome, and sperm head morphometry were seen compared to OVS.

Background

Approximately 40 to 50% of infertility in couples is male origin. Ten percent of this population belongs to azoospermic individuals. Sperm in these patients is obtained by various surgical methods [1]. Well-organized cryopreservation has some important advantages such as decreasing the risk of unnecessary ovulation induction, no need for synchronous oocyte retrieval, as well storing many vials for future use, especially in the case of the small testis, while damage from repeated testicular biopsies is avoided [2].

The sperm cryopreservation technique besides easily detectable damage, such as sperm motility, and morphology, can induce more subtle, sub-lethal sperm damage, following thawing. These damages can alter the composition of the spermatozoon’s plasma, acrosomal, and mitochondrial membranes, as well as the spermatozoon’s nucleus, chromatin decondensation, and induce DNA denaturation [3]. There is a concern about the proper cooling rate in these systems, especially regarding the preservation of the biopsied sperm from the testicles, due to the low number and greater vulnerability of these samples.

In oligozoospermia or azoospermia which are known as severe male factor infertility, if sperms can’t be found in the ejaculation, they must be earned through testicular biopsy specimens or epididymal sperm aspiration. In some cases, repeated sperm retrieval is needed which has many side effects, such as the economic burden, psychological pressure, and testicular damage [4]. Therefore, it is necessary to upgrade and improve existing techniques for the cryopreservation of sensitive spermatozoa.

In this regard, some efforts have been made by the researchers. In 1997, and 1998, when the zona pellucida was used as a freezing carrier for the few sperm [5, 6]. A few years later, the cryoloop was used for testicular and epididymal sperm cryopreservation [7]. Vitrification is performed in two distinguishable methods, “open” like cryoloop, and “closed” systems such as straw-in-straw, Cryotip, high-security vitrification straw, Cryopette, VitriSafe [8]. While samples are exposed to liquid nitrogen in the open systems, this is eliminated in the closed systems. Open systems are unsafe because of the disease transmission risk, but closed systems are safe [9]. In this regard when sperm cryopreservation (oligoasthenoteratozoospermia) was done using droplets, open-pulled straws, and open-standard straws, the researchers concluded that all three methods are good in the aim of sperm freezing, but, the open-pulled straw and open straw methods were preferred because of the isolation from liquid nitrogen, with a maximum reduction of the potential risk of microbial contamination [10]. According to reports, the cooling rate in open systems is more than – 20,000 °C/min, while is below – 2000 °C/min in closed systems [11]. A high percentage of the sperm population is unable to withstand the temperature changes induced during cryopreservation [12]. A small number of spermatozoa also was vitrified successfully, in a closed straw system, using two different methods of sperm loading: a spontaneous capillary action (CA), and a polar body biopsy (PBB) pipette [13].

Sperm and testicular tissue cryopreservation is capable of preserving male fertility for years. On the other hand, sperm head pathology may reflect genetic defects, which are related to abnormal early embryonic development or early pregnancy loss [14], so, finding a reliable method, and suitable device, which are accompanied by the lowest sperm structure changes, are of paramount importance. However, a reliable and safe protocol that can be used as a common technique for clinical cryopreservation of the few spermatozoa still has not been introduced.

It was mentioned that, both the conventional method of cryopreservation and present vitrification techniques are less suitable in cases in which the initial quality of semen is low, such as; severe oligozoospermia, cryptozoospermia, epididymal or testicular samples. A very limited number of studies have focused on the subject of cryopreservation of the small number of single spermatozoa, and more novel works are required, to improve sperm recovery after freezing. there`s some concern regarding the difficulties of such freezing protocols, as well as the toxic effect of permeable CPAs or the risk of cross-contamination in an open freezing system, [13]. For the first time, in this study, we aimed to systematically compare the efficacy of open (OVS) and closed (CVS) vitrification systems for freezing low sperm volumes, and assessing sperm motility, viability, and morphometric and chromatin characteristics. This study provides more insights into the optimal approach for single sperm cryopreservation and contributes novel findings to the existing literature.

Materials and methods

Sperm samples

This study was conducted in the Andrology clinic, at our infertility center, on 30 men with obstructive azoospermia (OA), who were referred to for diagnostic and therapeutic purposes. All patients (20 to 55 years) signed an institutional informed consent document, prior to participation. The unilateral or bilateral biopsy technique (testicular sperm extraction) was done routinely, under local anesthesia. Study samples were collected between January 2021 and January 2022.

After excision, the testicular tissue biopsies were placed into HEPES-buffered medium supplemented with human serum albumin (HSA, 5%). All earned surgical tissue samples were minced and dispersed using fine needles, in the Petry dish (10 cm). The tissue suspension was examined for the presence of sperm cells under high-power inverted microscopy. The testicular tissue and cellular suspensions were then centrifuged for 5 min at 300 × g, then 2 ml of erythrocyte lysing buffer was added to the testicular sperm pellet, 10 min at room temperature, and then washed by adding 5 ml of HAMs F10 medium, and centrifuged for another 10 min at 500 × g. Finally, the pellet product was re-suspended with 1.5 ml of culture media. If the sample contained the mature sperm sent for intra cytoplasmic sperm injection (ICSI), and the remaining went for vitrification.

For maturation induction, the testicular biopsied samples were cultured at 25 ̊C for 24 h after preparation [15]. After that sperm motility, DFI, and morphology were evaluated on the earned sample, according to the WHO manual [16], and sperm viability was assessed via emission of a single laser shot, as described later.

Single sperm vitrification procedure

A droplet of the prepared sample (1–3 μL) was transferred to the 5 μL droplet of Hams F10 (5% HSA), in the ICSI dish (Falcon, 1006 dishes) (RT). Twenty-five motile or sluggish spermatozoa with normal morphology were isolated using a homemade microinjection needle.

In the vitrified groups, a droplet of 0.3 µl CPAs (Vitrolife, Sweden) was placed on the polypropylene strip of a cryotech (Kitazato, Tokyo, Japan), at room temperature (Fig. 1) [17, 18]. The ICSI needle was placed into the narrow end of the cryotech and selected spermatozoa were then transferred into the cryoprotectant droplet. Since the post-thawed sperm motility in testicular cases was greatly reduced, Pentoxifylline was added to the extender [19].

Fig. 1
figure 1

a sperm freezing medium was loaded on the Cryotech device using a mouth pipet. b Testicular sperms were selected using an ICSI injection pipette and then transferred into the one droplet of sperm freezing medium

Full size image

In the end, for vitrification two methods were used; open (OVS) and closed (CVS) systems. For this purpose, after loading spermatozoa on the straws, in OVS, straws were immersed into LN2, without recaption of the straw, and after the vitrification, straws were caped in the LN2, before storage. In the CVS group, however, the straws were worn with plastic caps and then inserted into the other protective straws (similar to the straw-in-straw method). The protective straws were sealed before plunging into LN2. Subsequently, all the devices were exposed to LN2 directly for storage, and stored until warming.

After 7 days, for thawing, a 5-μL droplet of pre-warmed Hams F10 + 10% HSA was placed on a sterile ICSI dish. While the Cryotech was submerged in the LN2, the cap of cryotech was removed. Then, the tip of the cryotech containing sperms was quickly and directly placed in a pre-warmed droplet of Hams’ F10 medium supplemented with 5 mg/mL HSA. Thereafter the droplets containing sperm are covered by pre-warmed oil (Ovoil; Vitrolife, Sweden). Following 30 min incubation at 37 °C, the sperm cells were aspirated using the micropipette equipped with a micromanipulator on an inverted microscope and transferred to a new droplet of the basic medium [18].

Sperm parameters assessments

In the thawed sperm recovery evaluation, sperm motility and viability were assessed. For the motility assessment, under a phase-contrast microscope, any sign of sperm movement or twitching of the head or tail is considered as motile sperm and reported as total motility (%) rate. The Diff-Quik staining was used to assess the percentage of morphologically normal sperms (Fig. 2) (× 1000 magnification). The scoring was according to what was described in the WHO laboratory manual for sperm analysis [16].

Fig. 2
figure 2

Diff-quick staining, for sperm morphology scoring. a A normal shape sperm, b coiled tail after sperm warming, c sperm with misshaped head and excess residual cytoplasm (ERC), d sperm with amorphous head and broken tail in the weakened site, e proximal bent tail, f distal bent tail

Full size image

Sperm membrane integrity

As the spontaneous tail swellings take place after sperm cryopreservation, sperm viability was assessed using laser-assisted immotile sperm selection (LAISS). This system identifies the viable motile sperm via emission of a single laser shot of 129 μJ of approximately 1.2 Ms. Briefly if the tail coiled in response to the laser shot (OCTAX Laser Shot®, MTG, Germany), it would be considered viable. Besides this method is fast, simple, repeatable, and safe, there is no need for any chemical compounds for motility induction or flagellum curling [20].

Morphometric assessments

Knowing sperm characteristics and morphometric description can be effective in understanding the impact of cryopreservation on sperm cells (sperm cryo-tolerance), and is a useful method in the prediction of fertility. Measurements of the sperm head length and head width were performed using the MSOME (TE300; Nikon, Tokyo, Japan) and OCTAX software manually. The total viewing magnification of the spermatozoa on the computer monitor was × 6600 (Fig. 3). For each sperm head, the ellipticity, elongation, roughness, and regularity were calculated according to Table 1 [21, 22].

Fig. 3
figure 3

Measurements of sperm head length and head width were performed using the MSOME

Full size image
Table 1 Parameters assessed for the morphometric characterization of sperm heads
Full size table

Sperm chromatin assessment

Chromomycine A3 (CMA3) staining was performed as follows: recovered sperms were transferred on a glass slide and air-dried. Samples were fixed in Carnoy’s solution, stained by CMA3 solution for 10 min in the dark, and analyzed by fluorescence microscopy [23]. Bright yellow stained cells (chromatin packaging abnormal) were considered as CMA3+; while yellowish-green stained sperms (normal chromatin packaging) were considered as CMA3- (Fig. 4).

Fig. 4
figure 4

Sperm Chromomycine A3 staining (CMA3) staining, bright yellow stained cells (chromatin packaging abnormal) were considered as CMA3+; while yellowish-green stained sperms (normal chromatin packaging) were considered as CMA3-

Full size image

The sperm histone status was studied by aniline blue (AB) staining. Slides were fixed in a solution of 3% buffered glutaraldehyde stained with the AB stain for 5 min and assayed under a light microscope (Olympus, Tokyo, Japan). Unstained or pale-blue spermatozoa were considered normal mature, and dark-blue stained as abnormal spermatozoa (Fig. 5) [23].

Fig. 5
figure 5

The sperm histone status by aniline blue (AB) staining. Unstained or pale-blue spermatozoa were considered normal mature, and dark-blue stained as abnormal spermatozoa

Full size image

Sperm DNA fragmentation assessment

For the evaluation of the extent of DNA fragmentation, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was done using the In-Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany), following the manufacturer’s instructions. Spermatozoa with DNA fragmentation display a spectrum of green fluorescence and are considered TUNEL positive (Fig. 6) [24].

Fig. 6
figure 6

Extent of DNA fragmentation, (TUNEL) assay. Spermatozoa with DNA fragmentation showed yellow–red fluorescence (arrow), and sperm with intact DNA display green fluorescence (star)

Full size image

Acrosome integrity assessment

Briefly, spermatozoa were fixed with ethanol incubated with FITC-PSA (Sigma Co, USA), and evaluated using a fluorescent microscope. When over half of the sperm heads were bright and uniformly fluorescing, they were considered acrosome-intact. However, when the fluorescence band was found in the equatorial segment or any fluorescence staining in the acrosome area was considered acrosome-reacted (Fig. 7) [25].

Fig. 7
figure 7

Acrosome reaction test. When over half of the sperm heads were bright and uniformly fluorescing, they were considered as acrosome-intact. But when the fluorescence band was found at the equatorial segment or any fluorescence staining in the acrosome area was considered acrosome-reacted

Full size image

Statistical analysis

SPSS software was used for the evaluation of the effect of vitrification on sperm head morphometric dimensions. The data were analyzed by one-way analysis of variance (ANOVA) and expressed as means ± SD. The normal distribution of data was determined by the Kolmogorov–Smirnov normality test. The significance of differences between the groups was evaluated using the Tukey test and P < 0.05 was considered significant.

Results

Post-vitrification sperm characterizations

Overall, 1500 sperms were frozen on 60 straws (two straws, for each case). Fresh testicular samples (after 24 h culture) used in this study had the following characteristics: motility (76.3 ± 10.5), viability (76.8 ± 6.6%), and the other sperm parameters showed that the cryopreservation process greatly affected the sperm head characterizations, sperm DNA fragmentation, acrosome integrity and other morpho-kinematic parameters (Tables 2 and 3).

Table 2 The effect of vitrification systems of closed (CVS) and open (OVS) on the mean sperm morphometric parameters
Full size table
Table 3 The testicular sperm quality after vitrification in two systems (closed and opened) compare to fresh control group
Full size table

The sperm head dimensions in vitrified samples were smaller than those obtained in the fresh samples. Also, significant variations in width, perimeter, roughness, and regularity between fresh and post-thawing spermatozoa in the OVS group were detected (p < 0.05) (Table 2). Within analysis variation, the measurements for head length, ellipticity, and elongation were insignificant.

Motility, viability

The movement of biopsied spermatozoa before freezing was slow and sluggish (13.22 ± 3.6), even observed as twitching or wavy flagellar movement. Since the Post-thawed sperm motility in testicular cases was greatly reduced, Pentoxifylline was added to the thawing medium. The mean motility rate improved significantly and reached 76.3% in fresh spermatozoa. The sperm motility rate decreased significantly in both vitrified groups compared with the control group (16.14% and 6.06%, in CVS and OSV, respectively (p < 0.05)). The motility rate was higher in the closed compared with the open system (P = 0.0001).

Sperm viability revealed a mean value of 76.8% prior to vitrification. This value diminished to 54.8% in CVS and 47.1% in OVS after thawing (Table 3).

Sperm DNA integrity

The results for chromatin integrity (CMA3 +) showed that there was a significant reduction in normal intact chromatin structure after vitrification (29.25% in FC, vs. 32.37% in CVS, and 40.13% in OVS groups. On the other hand, significant impairment of sperm chromatin status was seen after the freeze and thawing procedure, with a high positive response to Aniline blue staining, in the vitrified samples.

In comparison to the fresh semen sample. Also, CVS has better results in post-thawed spermatozoa groups (AB + ; 30.37%, 37.92%, and 43.37%, respectively) (Table 3).

The sperm DNA fragmentation by TUNEL was 15.25% in the fresh control group and increased to 20.51% in the closed, and 23.17% in the open system group, after the freeze and thaw process (P = 0.0001) (Table 3).

Acrosome integrity

The results showed among the two cryopreservation methods OVS systems greatly affected acrosome integrity compared to the fresh control group (63.18% vs. 50.0% in the fresh group) (Table 3).

Sperm morphology

However, sperm morphology decreased during the freeze and thaw process, and the results were better in the closed system (p > 0.05) (Table 3).

Discussion

Sperm cryopreservation is an important procedure that is used in ART labs for different purposes [26]. Many studies have tried to find a safe method, containers, or better medium to increase sperm cryo-resistance, and the challenge of post-thaw sperm loss would be greater, when there is a low sperm count for freezing (such as severe oligozoospermia, or azoospermia), in the aim to increase ICSI success rate. During sperm freezing, the cell temperature decreases to − 196 °C and then will return to the body temperature during thawing. So, the cold and warm shocks can loss of normal sperm structure and functions.

The cryodamage in mammal spermatozoa is associated with head dimensions alteration [27]. In the current study, we also used the head’s measurements to evaluate which method of vitrification is associated with lower cryodamage, in the case of low semen volume; open, or closed system.

The results of this study showed more sperm head morphometry alteration, following vitrification with the OVS system, compared to the fresh control group. Since cryopreservation can cause sperm head alteration [28,29,30], it can be concluded that the close system (CVS) is a more optimal method, preserving more sperm head structure. Numerous mechanisms during cryopreservation may underlie the decrease in sperm head dimension, including osmotic changes [31], alterations of cell compartments [32], damage of the acrosome [33, 34], condensation of nuclear chromatin [35, 36], and sperm dehydration [37], while large sperm heads may be related to chromatin condensation defects, large nuclear pockets, and abnormal protamine ratios [14]. It was mentioned that sperm head morphology affects sperm fertilization capacity [38], and sperm head alteration is accompanied by a lower successful pregnancy after artificial insemination [29]. So, a safe and efficient sperm freezing method will be needed in the cases of OAT, which could guarantee sperm normality.

The results also showed a significant reduction in both sperm motility and viability, after vitrification. It is by other studies tried to vitrify single sperm in cases of severe oligozoospermia and azoospermia [39]. In this regard, better sperm motility and viability were earned, after the closed system compared to the open vitrification method. To the best of our knowledge, there has not been any study that investigated the cryo-tech device, in the two methods of open and closed system, for sperm freezing but there are some other studies that tried to cryopreserve of a small number of single spermatozoa in the open system [40, 41], or closed systems [17, 42, 43]. One of the main theories of these studies was the probability of microbial contamination with the open systems, and their results of using closed systems were satisfactory. Besides, some of the advantages of the closed devices, such as cryolock, and cryo-tech, are that allow for loading a very small volume of sperm (1–3 μL), and thawing the sperm samples directly into the ICSI dish, so there is no need for post-thawing washing and centrifugation [44].

More protamine deficiency and histone excess were observed in the OVS compared to CVS. Sperm is extraordinarily sensitive to rapid temperature reduction, which is called cold shock [45]. The protamine-histone replacement is a level of sperm nucleus organization that leads to condensation and compaction [46, 47]. The unbalanced protamine 1-to-protamine 2 ratios cause low chromatin compaction and higher DNA instability. These significantly enhance the likelihood of sperm DNA fragmentation [48].

The sperm DNA fragmentation increased after 24 h incubation, and also after the vitrification, especially in the open system. Other studies also showed that DNA fragmentation will be increased by the incubation and cryopreservation of the testicular spermatozoa [49], so for sperm cryopreservation, the method that is accompanied by minimal sperm DNA fragmentation should be used.

Some destructive events related to cold shock are intracellular enzymes and lipids releasing [50], also acrosomal and mitochondria membrane alterations (ion distribution change) may take place because of both osmotic and cold shock [51]. In this study, more intact acrosome was found following closed compared to open system. For successful fertilization, sperm must have a healthy plasma membrane, as well as acrosomes intact until insemination, for zona penetration and oocyte fusion [52]. So finding a reliable method for cryopreservation, which could guarantee the plasma and acrosome membrane, could increase the fertility rate of the warmed sperms.

Sperm morphology is the other parameter that declined after freezing [53]. However, the results were not significant, the morphological results were better in the closed system.

The osmolality and the tonicity of the sperm surrounding the medium are two main factors that influence sperm. During cryopreservation, the sperm membrane may change due to osmotic gradients [54], which can lead to the reduction of viability [55]. The reaction of spermatozoa to osmotic challenge and their ability to regulate cell volume can determine sperm cryopreservability [56]. Almost all components of the membrane suffer from the freeze and thaw process. The rate of these changes is different among species and depends on the arrangement and biophysical properties of the plasma membrane [57]. A decrease in sperm viability after thawing, especially in OVS, shows that the membrane has undergone noticeable changes.

In addition, our observation elucidates the loss of acrosome during the vitrification process. According to a hypothesis, the cytoplasmatic Ca+2 levels can increase along with lowering temperature which ends up in a capacitated-like reaction, ion leakage, and exocytosis of acrosomal contents [58]. The decline of intact acrosome after cryopreservation may be the reason for the reduced reproductive potential of human spermatozoa. Also, an increase in the size of sperm components, as well as sperm head elongation is associated with better motility [59]. According to this theory, for more energy sources, the spermatozoa with larger heads swim faster. The decrease in sperm motility occurred following the vitrification. Perhaps, one of the causes of this lesion is the morphological changes in the sperm head.

In the CVS state with the falling of the cooling rate, the sperm alterations were less, and the samples were not in direct contact with liquid nitrogen. According to the other previous studies, it was shown that vitrification using a closed system does not compromise sperm motility, while in the double straw design, the specimen is not exposed to LN2 [60].

Conclusion

The recovered testicular spermatozoa from CVS showed much higher sperm viability and fewer alterations in the chromatin, acrosome, and sperm head morphometry than OVS. As sperm head pathology, as a marker of genetic defects, increased after cryopreservation, the current findings are behind the safety of CVS with the lower head parameter changes.

Availability of data and materials

Derived data supporting the findings of this study are available from https://doi.org/10.7910/DVN/EPX623

References

  1. Huang C, Gan RX, Hu JL, Liu F, Hong Y, Zhu WB et al (2022) Clinical benefit for cryopreservation of single human spermatozoa for ICSI: a systematic review and meta-analysis. Andrology 10:82–91

    Article  PubMed  CAS  Google Scholar 

  2. Karacan M, Alwaeely F, Erkan S, Çebi Z, Berberoğlugil M, Batukan M et al (2013) Outcome of intracytoplasmic sperm injection cycles with fresh testicular spermatozoa obtained on the day of or the day before oocyte collection and with cryopreserved testicular sperm in patients with azoospermia. Fertil Steril 100:975–980

    Article  PubMed  Google Scholar 

  3. Boitrelle F, Albert M, Theillac C, Ferfouri F, Bergere M, Vialard F et al (2012) Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. J Androl 33:1371–1378

    Article  PubMed  Google Scholar 

  4. Ishikawa T (2012) Surgical recovery of sperm in non-obstructive azoospermia. Asian JAndrol 14:109

    Article  Google Scholar 

  5. Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT (1997) Cryopreservation of single human spermatozoa. Hum Reprod 12:994–1001

    Article  PubMed  CAS  Google Scholar 

  6. Desai N (1998) A convenient technique for cryopreservation of micro quantities of sperm. Fertil Steril. S197–S8.

  7. Desai NN, Blackmon H, Goldfarb J (2004) Single sperm cryopreservation on cryoloops: an alternative to hamster zona for freezing individual spermatozoa. Reprod Biomed Online 9:47–53

    Article  PubMed  Google Scholar 

  8. Tao Y, Sanger E, Saewu A, Leveille M-C (2020) Human sperm vitrification: the state of the art. Reprod Biol Endocrinol 18:1–10

    Article  Google Scholar 

  9. Vajta G, Rienzi L, Ubaldi FM (2015) Open versus closed systems for vitrification of human oocytes and embryos. Reprod Biomed Online 30:325–333

    Article  PubMed  Google Scholar 

  10. Isachenko V, Isachenko E, Montag M, Zaeva V, Krivokharchenko I, Nawroth F et al (2005) Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online 10:350–354

    Article  PubMed  Google Scholar 

  11. AbdelHafez F, Xu J, Goldberg J, Desai N (2011) Vitrification in open and closed carriers at different cell stages: assessment of embryo survival, development, DNA integrity and stability during vapor phase storage for transport. BMC Biotechnol 11:29

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Watson PF (2000) The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 60–61:481–492

    Article  PubMed  Google Scholar 

  13. Kuznyetsov V, Moskovtsev SI, Crowe M, Lulat AG-M, Librach CL (2015) Vitrification of a small number of spermatozoa in normozoospermic and severely oligozoospermic samples. Syst Biol Reprod Med 61:13–17

    Article  PubMed  Google Scholar 

  14. Yurchuk T, Petrushko M, Gapon A, Piniaiev V, Kuleshova L (2021) The impact of cryopreservation on the morphology of spermatozoa in men with oligoasthenoteratozoospermia. Cryobiology 100:117–124

    Article  PubMed  CAS  Google Scholar 

  15. Hosseini A, Khalili MA (2017) Improvement of motility after culture of testicular spermatozoa: the effects of incubation timing and temperature. Transl Androl Urol 6:271

    Article  PubMed  PubMed Central  Google Scholar 

  16. Organization WH (2010) WHO laboratory manual for the examination and processing of human semen.

  17. Endo Y, Fujii Y, Shintani K, Seo M, Motoyama H, Funahashi H (2012) Simple vitrification for small numbers of human spermatozoa. Reprod Biomed Online 24:301–307

    Article  PubMed  CAS  Google Scholar 

  18. Hosseini A, Khalili MA, Talebi AR, Agha-Rahimi A, Ghasemi-Esmailabad S, Woodward B et al (2018) Cryopreservation of Low Number of Human Spermatozoa; Which is Better: Vapor Phase or Direct Submerging in Liquid Nitrogen? Hum Fertil. 22:1–7

    CAS  Google Scholar 

  19. Nabi A, Khalili MA, Fesahat F, Talebi A, Ghasemi-Esmailabad S (2017) Pentoxifylline increase sperm motility in devitrified spermatozoa from asthenozoospermic patient without damage chromatin and DNA integrity. Cryobiology 76:59–64

    Article  PubMed  CAS  Google Scholar 

  20. Nordhoff V (2015) How to select immotile but viable spermatozoa on the day of intracytoplasmic sperm injection? An embryologist’s view. Andrology 3:156–162

    Article  PubMed  CAS  Google Scholar 

  21. Soler C, Contell J, Bori L, Sancho M, García-Molina A, Valverde A et al (2017) Sperm kinematic, head morphometric and kinetic-morphometric subpopulations in the blue fox (Alopex lagopus). Asian J Androl 19:154

    Article  PubMed  Google Scholar 

  22. Cursino MS, Duarte JMB (2016) Using sperm morphometry and multivariate analysis to differentiate species of gray Mazama. R Soc Open Sci 3:160345

    Article  PubMed  PubMed Central  Google Scholar 

  23. Talebi A, Vahidi S, Aflatoonian A, Ghasemi N, Ghasemzadeh J, Firoozabadi R et al (2012) Cytochemical evaluation of sperm chromatin and DNA integrity in couples with unexplained recurrent spontaneous abortions. Andrologia 44:462–470

    Article  PubMed  Google Scholar 

  24. Lusignan M-F, Li X, Herrero B, Delbès G, Chan PT (2018) Effects of different cryopreservation methods on DNA integrity and sperm chromatin quality in men. Andrology 6:829–835

    Article  PubMed  CAS  Google Scholar 

  25. Samavat J, Natali I, Degl’Innocenti S, Filimberti E, Cantini G, Di Franco A et al (2014) Acrosome reaction is impaired in spermatozoa of obese men: a preliminary study. Fertil Steril 102:1274–81.e2

    Article  PubMed  CAS  Google Scholar 

  26. Gómez-Torres MJ, Medrano L, Romero A, Fernández-Colom PJ, Aizpurúa J (2017) Effectiveness of human spermatozoa biomarkers as indicators of structural damage during cryopreservation. Cryobiology. 78:90–4

    Article  PubMed  Google Scholar 

  27. Esteso MC, Soler AJ, Fernandez-Santos MR, Quintero-Moreno AA, Garde JJ (2006) Functional significance of the sperm head morphometric size and shape for determining freezability in iberian red deer (Cervus elaphus hispanicus) epididymal sperm samples. J Androl 27:662–670

    Article  PubMed  Google Scholar 

  28. Cerdeira J, Sánchez-Calabuig MJ, Pérez-Gutiérrez JF, Hijon M, Castaño C, Santiago-Moreno J (2020) Cryopreservation effects on canine sperm morphometric variables and ultrastructure: Comparison between vitrification and conventional freezing. Cryobiology 95:164–170

    Article  PubMed  CAS  Google Scholar 

  29. Hidalgo M, Rodríguez I, Dorado JM (2007) The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability. Anim Reprod Sci 100:61–72

    Article  PubMed  CAS  Google Scholar 

  30. Esteso MC, Fernández-Santos MR, Soler AJ, Montoro V, Quintero-Moreno A, Garde JJ (2006) The effects of cryopreservation on the morphometric dimensions of Iberian red deer (Cervus elaphus hispanicus) epididymal sperm heads. Reprod Domest Anim 41:241–246

    Article  PubMed  CAS  Google Scholar 

  31. Curry M, Kleinhans F, Watson P (2000) Measurement of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped fluorophore. Cryobiology 41:167–173

    Article  PubMed  CAS  Google Scholar 

  32. Xin M, Niksirat H, Shaliutina-Kolešová A, Siddique MAM, Sterba J, Boryshpolets S et al (2020) Molecular and subcellular cryoinjury of fish spermatozoa and approaches to improve cryopreservation. Rev Aquac 12:909–924

    Article  Google Scholar 

  33. Thurston LM, Watson PF (2002) Semen cryopreservation: a genetic explanation for species and individual variation? CryoLetters 23:255–262

    PubMed  Google Scholar 

  34. Villaverde-Morcillo S, Esteso M, Castaño C, Toledano Díaz A, López-Sebastián A, Campo J et al (2015) Influence of staining method on the values of avian sperm head morphometric variables. Reprod Domest Anim 50:750–755

    Article  PubMed  CAS  Google Scholar 

  35. Álvarez M, García-Macías V, Martínez-Pastor F, Martínez F, Borragán S, Mata M et al (2008) Effects of cryopreservation on head morphometry and its relation with chromatin status in brown bear (Ursus arctos) spermatozoa. Theriogenology 70:1498–1506

    Article  PubMed  Google Scholar 

  36. Rijsselaere T, Van Soom A, Hoflack G, Maes D, de Kruif A (2004) Automated sperm morphometry and morphology analysis of canine semen by the Hamilton-Thorne analyser. Theriogenology 62:1292–1306

    Article  PubMed  Google Scholar 

  37. Sieme H, Oldenhof H, Wolkers W (2015) Sperm membrane behaviour during cooling and cryopreservation. Reprod Domest Anim 50:20–26

    Article  PubMed  CAS  Google Scholar 

  38. Gangwar C, Saxena A, Shukla P, Singh S, Patel A, Antil M et al (2019) Cryopreservation Induced Alteration in SpermMorphology in Hariana Bull Semen. 9:282–288

    Google Scholar 

  39. Maleki B, Khalili MA, Gholizadeh L, Mangoli E, Agha-Rahimi A (2022) Single sperm vitrification with permeable cryoprotectant-free medium is more effective in patients with severe oligozoospermia and azoospermia. Cryobiology 104:15–22

    Article  PubMed  CAS  Google Scholar 

  40. Endo Y, Fujii Y, Shintani K, Seo M, Motoyama H, Funahashi H (2011) Single spermatozoon freezing using Cryotop. J Mammalian Ova Res 28:47–52

    Article  Google Scholar 

  41. Chen Y, Li L, Qian Y, Xu C, Zhu Y, Huang H et al (2015) Small-volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop. Andrologia 47:694–699

    Article  PubMed  CAS  Google Scholar 

  42. Luyet BJ, Hodapp EL (1938) Revival of frog’s spermatozoa vitrified in liquid air. Proc Soc Exp Biol Med 39:433–434

    Article  Google Scholar 

  43. Herrler A, Eisner S, Bach V, Weissenborn U, Beier HM (2006) Cryopreservation of spermatozoa in alginic acid capsules. Fertil Steril 85:208–213

    Article  PubMed  CAS  Google Scholar 

  44. Stein A, Shufaro Y, Hadar S, Fisch B, Pinkas H (2015) Successful use of the Cryolock device for cryopreservation of scarce human ejaculate and testicular spermatozoa. Andrology 3:220–224

    Article  PubMed  CAS  Google Scholar 

  45. Abedin SN, Baruah A, Baruah KK, Bora A, Dutta DJ, Kadirvel G et al (2023) Zinc oxide and selenium nanoparticles can improve semen quality and heat shock protein expression in cryopreserved goat (Capra hircus) spermatozoa. J Trace Elem Med Biol 80:127296

    Article  PubMed  CAS  Google Scholar 

  46. Kipper BH, Trevizan JT, Carreira JT, Carvalho IR, Mingoti GZ, Beletti ME et al (2017) Sperm morphometry and chromatin condensation in Nelore bulls of different ages and their effects on IVF. Theriogenology 87:154–160

    Article  PubMed  CAS  Google Scholar 

  47. Ward WS, Coffey DS (1991) DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod 44:569–574

    Article  PubMed  CAS  Google Scholar 

  48. Gosálvez J, López-Fernández C, Fernández JL, Gouraud A, Holt WV (2011) Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species. Mol Reprod Dev 78:951–961

    Article  PubMed  Google Scholar 

  49. Dalzell LH, McVicar CM, McClure N, Lutton D, Lewis SEM (2004) Effects of short and long incubations on DNA fragmentation of testicular sperm. Fertil Steril 82:1443–1445

    Article  PubMed  Google Scholar 

  50. Akhter S, Rakha BA, Ansari MS, Husna AU, Iqbal S, Khalid M (2017) Evaluation of quail and turkey egg yolk for cryopreservation of Nili-Ravi buffalo bull semen. Theriogenology 87:259–265

    Article  PubMed  CAS  Google Scholar 

  51. Ba BALL, Vo A (2001) Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. J Androl 22:1061–1069

    Article  Google Scholar 

  52. Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA et al (2011) Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization. Proc Natl Acad Sci 108:4892–4896

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Le MT, Nguyen TTT, Nguyen TT, Nguyen VT, Nguyen TTA, Nguyen VQH et al (2019) Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects. Eur J Obstet Gynecol Reprod Biol 234:14–20

    Article  PubMed  CAS  Google Scholar 

  54. Hammerstedt RH, Graham JK, Nolan JP (1990) Cryopreservation of mammalian sperm: what we ask them to survive. J Androl 11:73–88

    Article  PubMed  CAS  Google Scholar 

  55. Mohamed MSA (2015) Slow cryopreservation is not superior to vitrification in human spermatozoa; an experimental controlled study. Iran J Reprod Med 13:633

    Google Scholar 

  56. García-Herreros M, Barón F, Aparicio I, Santos A, García-Marín L, Gil M (2008) Morphometric changes in boar spermatozoa induced by cryopreservation. Int J Androl 31:490–498

    Article  PubMed  Google Scholar 

  57. Parks J, Graham J (1992) Effects of cryopreservation procedures on sperm membranes. Theriogenology 38:209–222

    Article  PubMed  CAS  Google Scholar 

  58. Silva P, Gadella B (2006) Detection of damage in mammalian sperm cells. Theriogenology 65:958–978

    Article  PubMed  CAS  Google Scholar 

  59. Villaverde-Morcillo S, Soler A, Esteso M, Castaño C, Miñano-Berna A, Gonzalez F et al (2017) Immature and mature sperm morphometry in fresh and frozen-thawed falcon ejaculates. Theriogenology 98:94–100

    Article  PubMed  CAS  Google Scholar 

  60. Kuleshova L, Shaw JM (2000) A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen. Hum Reprod 15:2604–2609

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to thank all the participants in this study. Also, we appreciate Fatemeh Yazdinejad for providing the samples, and for technical help.

Funding

There is no funding.

Author information

Authors and Affiliations

  1. Research and Clinical Center for Infertility, Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

    Mohammad Ali Khalili & Esmat Mangoli

  2. Molecular Medicine Research Center, Research Institute of Basic Medical Sciences, Rafsanjan University of Medical Sciences, Rafsanjan, Iran

    Mahboubeh Vatanparast

  3. Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran, University of Medical Sciences, Tehran, Iran

    Saeed Ghasmi-Esmaeilabad

  4. Najmiyeh Infertility Treatment Center, Kerman, Iran

    Mojgan Moshrefi

  5. Department of Anatomical Sciences and Reproductive Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

    Akram Hosseini

Authors
  1. Mohammad Ali Khalili
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mahboubeh Vatanparast
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Esmat Mangoli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Saeed Ghasmi-Esmaeilabad
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mojgan Moshrefi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Akram Hosseini
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M A Khalili contributed to research design and manuscript drafting and approval. M Vatanparast contributed to manuscript revision and approval. E Mangoli and Saeid Ghasmi Esmaeilabadi performed the research. M Moshrefi contributed to data collection and analysis. Akram Hosseini interpreted the data and helped revise and approve the manuscript. All authors read and approve the final manuscript.

Corresponding authors

Correspondence to Mahboubeh Vatanparast or Akram Hosseini.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Yazd Reproductive Sciences Institute, which follows the Helsinki Declaration of 1975 (Ir.ssu.rsi.rec.1394.30).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Khalili, M.A., Vatanparast, M., Mangoli, E. et al. Comparison between open and closed systems for vitrification of individual sperm: assessing morphometric measurements and chromatin integrity. Middle East Fertil Soc J 29, 39 (2024). https://doi.org/10.1186/s43043-024-00188-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s43043-024-00188-4

Keywords