1. Introduction
Human sperm cryopreservation is a technique used routinely in assisted reproductive technologies (ART) clinics in order to preserve male fertility in case the patient is undergoing a procedure that may affect his fertility, such as chemo- or radiotherapy and vasectomy ( 1 ). This technique is also used in the case of sperm donors to prevent the transition of infectious diseases ( 2 ). It is approved that the quality of sperm significantly decreases after cryopreservation (post-thawing stage), which is due to the formation of ice crystals and the changes that occur in the sperm plasma membrane ( 3 ). The success of sperm cryopreservation is measured by the percentage of the motile sperm recuperated at the post-thawing stage. Based on several studies, about 30-40% of the motility of the sample is lost at the post-thawing stage. This is due to the structural damage of the plasma membrane, acrosomal contents, and DNA integrity ( 4 - 6 ).
Not only ejaculated sperms can be cryopreserved but also epidydimal and testicular sperms can be retrieved and then cryopreserved. The main reason for epidydimal or testicular aspiration is obstructive azoospermia; therefore, the amount of semen that is obtained in this technique is relatively few ( 7 ).
The conventional sperm cryopreservation techniques are not appropriate for samples from severe oligozoospermic or obstructive azoospermic patients since the sperms are diluted and lost during centrifugation ( 8 , 9 ). For this reason, several researchers have tried to develop a proper technique to cryopreserve a low number of sperms to replace the conventional sperm cryopreservation techniques in order to preserve the low number of sperms obtained from oligozoospermic or obstructive azoospermic patients ( 5 , 9 - 13 ).
This study aimed to preserve the low concentration of cryopreserved sperms at the post-thawing stage by using emptied sheep ovarian follicles as a carrier for sperms during cryopreservation.
2. Materials and Methods
2.1. Subjects and Samples Collection
In this study, 30 semen samples were collected from oligozoospermic patients (sperm concentration ≤15×106 mL-1) and 10 samples from non-oligozoospermic males (sperm concentration 16-20×106 mL-1) within the age range of 19-54 years old. All semen samples were collected from the subjects by masturbation after 3 days of sexual abstinence. The samples were analyzed according to the guidelines of the World Health Organization 2010 ( 14 ). using a light microscope (Optica, Italy) to determine the sperm parameters (sperm concentration, total motility, progressive motility, and normal morphology). Each sample was analyzed twice by only one experienced biologist to avoid personal variations and ensure accurate and precise results.
2.2. Experimental Design
After seminal fluid analysis, 40 samples were equally divided into four groups (G1-G4) according to the sperm concentration (10 samples in each group), and each sample was divided into two equal parts. One part was diluted 1:1 with a simple medium for assisted reproductive technologies (SMART) medium ( 15 ) and injected into the emptied ovarian follicles of sheep without the addition of any kind of cryoprotectant. Besides, the other part was diluted 1:1 with cryosolution that contained 10% glycerol and injected inside the emptied ovarian follicles for cryopreservation.
2.3. Preparation of Cryoprotectant
The glycerol-based cryosolution was prepared by adding 1 mL (10%) of glycerol to 9 mL of SMART medium.
2.4. Collection and Preparation of Sheep Ovarian Follicles
In total, 480 ovarian follicles were sliced from the ovaries of 425 sheep in this study. The sheep ovaries were collected from a local slaughterhouse. Both ovaries were collected directly from the ewes after they were slaughtered and kept at 32-35 ºC with normal saline solution (0.9% NaCl) supplemented with two types of antibiotics (100 mg/ml penicillin and 100 mg/ml streptomycin). Afterward, the ovaries were transported to the laboratory within 1 h. In the laboratory, ovaries were washed three times using a normal saline solution (37 ºC) to remove the clotted blood and reduce contamination on the ovarian surface ( 16 ).
After washing, the ovaries differentiated according to the size of the ovarian follicles. The ovaries that contained follicles less than 0.3 mm in diameter were excluded and those that contained follicles larger than 0.3 mm in size were sliced to remove the medulla and allow the follicles to fit inside the cryotube. Subsequently, the ovarian pieces that contained the follicles were stored at 4˚C till the semen was prepared.
2.5. Process of Sperm Cryopreservation
The follicular fluid was extracted from the prepared ovarian follicles using a 23-gage sterile hypodermic needle with a disposable 2 mL syringe. Afterward, each part of the semen samples was injected into four emptied follicles and inserted into 1.8 mL cryotubes (Thermo-Scientific), and covered with cryosolution. The cryotube was then exposed to liquid nitrogen (LN2) vapor for 15 min (2 cm above the surface of LN2). Finally, the cryotubes were plunged inside LN2 and stored at -196 °C for 2 months using a cryopreservation LN2 tank (MVE SC series LN2 tank of 40 L).
2.6. Thawing Process
After two months of cryopreservation, each cryotube was extracted from the LN2 and immersed inside a water bath at 35 °C for 5 min. Afterward, by using forcipes, the ovarian follicles were transferred from the cryotube to a clean Petri dish and the samples were withdrawn from the follicles using a 23-gage sterile hypodermic needle with a disposable 3 mL syringe. The volume was measured and the samples that were cryopreserved with glycerol were diluted 1:1 volume with the thawing solution (SMART medium plus 0.2 M sucrose) to remove the glycerol and the samples that cryopreserved without cryoprotectant were not diluted. Afterward, the sperm parameters (sperm concentration, motility, and normal morphology) were measured for all samples.
2.7. Statistical Analysis
The statistical analysis system (SAS) software (2012) was used to analyze the data. Parameters in this study were expressed as the mean and standard deviation (mean±SD) and the recovery rate of groups at the post-thawing stage was expressed as a percentage. The least significant difference test and analysis of variance were used to analyze the differences among groups. The P values of less than 0.01 were considered statistically significant.
3. Results
Sperm concentration was significantly (P<0.01) decreased at the post-thawing stage, compared to the pre-freezing stage for all the studied groups. Moreover, in all groups, the sperm concentration significantly (P<0.01) increased in samples that were cryopreserved without cryoprotectant, compared to those that were cryopreserved with glycerol (Table 1). About 50% of the sperm concentration was recovered at the post-thawing stage in samples cryopreserved without cryoprotectant, while about 25% of sperm concentration was recovered in samples that were cryopreserved with glycerol (Figure 1a).
| Group (☓ 106 mL-1) | Fresh | CF | Gly 10% |
|---|---|---|---|
| G1: (3-5) | 3.98±0.72a | 1.96±0.21b | 0.95±0.11c |
| G2: (6-10) | 7.52±1.48a | 3.74±1.09b | 1.88±0.05c |
| G3: (11-15) | 12.65±1.37a | 6.30±0.89b | 2.98±0.18c |
| G4: (16-20) | 17.77±1.35a | 8.86±1.16b | 4.40±0.32c |
| Data are presented as the mean ±SD. Fresh: fresh semen pre-freezing, CF: cryoprotectant-free cryopreserved sperms, Gly: glycerol. Different small letters (a, b, and c) indicate significant differences (P<0.01) in the same row | |||
Figure 1. Recovery rates (%) of human sperm parameters for all study groups after freezing-thawing using sheep’s ovarian follicles. (a) sperm concentration, (b) progressive motility, (c) total motility, (d) normal morphology. Different letters on the percentage bars indicate significant differences (P<0.01)
Progressive sperm motility and total motility also significantly (P<0.01) decreased at the post-thawing stage in all the studied groups, compared to the pre-freezing stage. In all groups, the samples that were cryopreserved with glycerol showed a significant (P<0.01) increase in progressive and total motility, compared to those that were cryopreserved without cryoprotectant (Tables 2 and 3). The highest and lowest progressive motility recovery rates were observed in groups four and one (Figure 1b), respectively. Moreover, the highest total motility recovery rate was observed in groups three and four, while the lowest was observed in groups one and two (Figure 1c).
| Group (☓ 106 mL-1) | Fresh | CF | Gly 10% |
|---|---|---|---|
| G1: (3-5) | 30.25±4.65a | 12.46±0.21b | 16.27±1.76c |
| G2: (6-10) | 26.22±5.18a | 11.98±1.09b | 14.75±2.54c |
| G3: (11-15) | 23.10±5.28a | 10.60±1.89b | 13.17±1.05c |
| G4: (16-20) | 22.90±9.52a | 11.86±4.25b | 15.75±3.04c |
| Data are presented as the mean±SD. Fresh: fresh semen pre-freezing, CF: cryoprotectant-free cryopreserved sperms, Gly: glycerol. Different small letters (a, b, and c) indicate significant differences (P<0.01) in the same row | |||
| Group (☓ 106 mL-1) | Fresh | CF | Gly 10% |
|---|---|---|---|
| G1: (3-5) | 46.53±9.30a | 25.21±4.76b | 29.27±3.46c |
| G2: (6-10) | 34.92±6.25a | 18.48±4.46b | 22.67±3.98c |
| G3: (11-15) | 32.55±6.29a | 19.14±3.96b | 23.86±2.67c |
| G4: (16-20) | 33.80±7.37a | 20.86±4.65b | 24.95±4.23c |
| Data are presented as the mean ±SD. Fresh: fresh semen pre-freezing, CF: cryoprotectant-free cryopreserved sperms, Gly: glycerol. Different small letters (a, b, and c) indicate significant differences (P<0.01) in the same row | |||
Normal sperm morphology (%) did not differ significantly (P<0.01) neither between pre-and post-thawing stages nor between post-thawing cryoprotectant and cryoprotectant-free treats in all studied groups (Table 4). The best recovery rate of normal morphology was found in samples that were cryopreserved with glycerol in group three (97.58%). However, the lowest recovery rate of normal morphology was observed in samples that were cryopreserved without cryoprotectants in group four (91.99%) (Figure 1d).
| Group (☓ 106 mL-1) | Fresh | CF | Gly 10% |
|---|---|---|---|
| G1: (3-5) | 40.21±1.14Aa | 38.23±1.31 Aa | 38.32±1.37Aa |
| G2: (6-10) | 10.52±5.75Ca | 9.76±2.79 Da | 9.93±3.31 Da |
| G3: (11-15) | 23.16±3.79Ba | 21.60±1.05 Ba | 22.60±1.35Ba |
| G4: (16-20) | 20.35±3.01Ba | 18.72±1.81 Ca | 19.03±1.41Ca |
| Data are presented as the mean ±SD. Fresh: fresh semen pre-freezing, CF: cryoprotectant-free cryopreserved sperms, Gly: glycerol. Different small letters (a, b, and c) indicate significant differences (P<0.01) in the same row | |||
4. Discussion
In this study, the emptied ovarian follicles of sheep were used as a carrier for human sperm during cryopreservation. It was tried to cryopreserve sperms in this carrier without using cryoprotectant to avoid the loss of sperms through dilutions and washing steps. The results were compared with those of the studies that used glycerol as a cryoprotectant in the same technique. Consequently, different concentrations of sperm were successfully cryopreserved by this technique.
Previously, Cohen, Garrisi ( 10 ) described the emptied human and mouse zona pellucida (ZP) as a vehicle to cryopreserve single human spermatozoa. Cohen’s technique was upgraded by Hsieh, Tsai ( 9 ) to cryopreserve more than one sperm per ZP. In the same concept, Just, Gruber ( 17 ) used the spherical algae Volvox globator and they succeeded to cryopreserve 15-18 sperms per each spherical Volvox. However, these techniques cryopreserved a limited number of sperms and required time to select the sperms by a micromanipulator. In the present study, the sperms were injected with their seminal plasma (hole semen) directly inside the emptied ovarian follicles.
This technique is an easier and non-time consumer one that also does not require micromanipulation, like previous techniques. The ovarian follicle of sheep is easy to obtain from any slaughterhouse and cost-efficient also its size is suitable for carrying about 0.2-0.5 mL of semen which allows cryopreserving an unlimited number of sperms (depending on the original sperm concentration within semen). The retrieval of the cryopreserved sperms from the follicles at the post-thawing stage is also simple and does not need enzymatic aids. The proteins in the seminal plasma protect the spermatozoa during the attachment between spermatozoa and the granulosa cells of the follicular wall and make the probability of acrosome reaction not anticipated.
In samples that were cryopreserved without cryoprotectant, the semen was diluted 1:1 with SMART medium to reach the volume as that of the semen cryopreserved with glycerol. Meanwhile, samples that were cryopreserved with glycerol were diluted again at the post-thawing stage with the thawing solution to remove the glycerol. Therefore, the recovery of sperm concentration at the post-thawing stage was ~50% for samples cryopreserved without cryoprotectant and ~25% for samples cryopreserved with glycerol in all groups. Cryopreservation of sperms in this technique without using cryoprotectant did not require the dilution of the sample; therefore, the concentration recovery rate at the post-thawing stage will be about 98-100% and this will be a good choice for cases of severe oligozoospermia.
The post-thawing progressive motility and total motility in samples cryopreserved without cryoprotectant were significantly lower, compared to that in samples cryopreserved with glycerol. However, the recovery rate of progressive and total motility in samples cryopreserved without cryoprotectant was hopeful. These promising results reflect the ability of this technique to recover sperm motility at the post-thawing stage without the use of cryoprotectant. Several factors may participate in the protection of sperm viability and support sperm motility, including the presence of granulosa cells, follicular wall, some biochemical components of seminal plasma, and the technique of sperm processing and freezing.
The best recovery rate in sperm motility was obtained by the use of glycerol in all studied groups and this came along with previous publications that mentioned the importance of cryoprotectants and their impact on cryopreservation outcomes ( 18 , 19 ). It was found that the recovery rate in progressive and total motility increased along with the sperm concentration (the lowest and highest in groups one and four, respectively) and this may refer to an association between sperm concentration and motility recovery at the post-thawing stage. Zhang, Zhou ( 20 ) investigated the effect of pre-freezing conditions on the progressive motility recovery rate. They found a significant association between sperm concentration and progressive motility at the pre-freezing stage with the progressive motility recovery rate, which is in concordance with the results of the present study in this regard.
In the present study, the normal sperm morphology was not significantly affected during cryopreservation with or without cryoprotectant unlike the progressive motility and total motility. It has been declared previously that about 50% of sperms cannot survive cryopreservation even with optimized protocols ( 21 ). Le, Nguyen ( 22 ) stated that cryopreservation has potential effects on sperm motility, viability, and morphology. The findings of the present research indicated that the harmful effect of freezing in this technique was restricted to the plasma membrane which led to the reduction of the motility more than normal morphology.
In conclusion, ovarian follicles of emptied sheep are an ideal carrier for cryopreservation of different sperm concentrations and it is suitable for cryopreservation of low concentrations of sperms. In this technique, sperms can be successfully cryopreserved without using cryoprotectants; however, the best motility recovery rate can be obtained by using glycerol-based cryosolution. Further studies are required to confirm these results, investigate the outcomes of cryopreserved testicular and epididymal sperms, and evaluate the fertility rate of cryopreserved sperms in this technique.
Authors' Contribution
Study concept and design: A. H. Z.
Acquisition of data: A. H. Z.
Analysis and interpretation of data: M. B. M. R. K.
Drafting of the manuscript: H. H. H.
Critical revision of the manuscript for important intellectual content: A. H. Z.
Statistical analysis: M. B. M. R. K.
Administrative, technical, and material support: H. H. H.
Ethics
Informed consent was obtained from all the participants before the collection of semen samples. Moreover, the study was ethically approved by the local medical Ethics Committee (Approval No: 19-0001).
Conflict of Interest
The authors declare that they have no conflict of interest.
References
- Pabon D, Meseguer M, Sevillano G, Cobo A, Romero JL, Remohi J, et al. A new system of sperm cryopreservation: evaluation of survival, motility, DNA oxidation, and mitochondrial activity. Andrology. 2019; 7(3):293-301.
- Mocé E, Fajardo A, Graham J. Human Sperm cryopreservation. Eur Med J. 2016; 1(1):86-91.
- Shahba MI, El-Sheshtawy RI, El-Azab A-SI, Abdel-Ghaffar AE, Ziada MS, Zaky AA. The effect of freeze-drying media and storage temperature on ultrastructure and DNA of freeze-dried buffalo bull spermatozoa. Asian Pac J Reprod. 2016; 5(6):524-35.
- Boitrelle F, Albert M, Theillac C, Ferfouri F, Bergere M, Vialard F, et al. Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. J Androl. 2012; 33(6):1371-8.
- Hatakeyama S, Tokuoka S, Abe H, Araki Y, Araki Y. Cryopreservation of very low numbers of spermatozoa from male patients undergoing infertility treatment using agarose capsules. Hum Cell. 2017; 30(3):201-8.
- Varghese A, Nandi P, Mahfouz R, Athayde K, Agarwal A. Human sperm cryopreservation. Androl Lab Man. 2010; 1:196-206.
- Elnaser T, Rashwan H. Testicular sperm extraction and cryopreservation in patients with non-obstructive azoospermia prior to ovarian stimulation for ICSI. Middle East Fertil Soc J. 2004; 9(2):128-35.
- Di Santo M, Tarozzi N, Nadalini M, Borini A. Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART. Adv Urol. 2012; 2012
- Hsieh Y, Tsai H, Chang C, Lo H. Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae. Fertil Steril. 2000; 73(4):694-8.
- Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT. Cryopreservation of single human spermatozoa. Hum Reprod. 1997; 12(5):994-1001.
- Desai NN, Blackmon H, Goldfarb J. Single sperm cryopreservation on cryoloops: an alternative to hamster zona for freezing individual spermatozoa. Reprod Biomed Online. 2004; 9(1):47-53.
- Sohn JO, Jun SH, Park LS, Kim EK, Chung TG, Lee DR. Comparison of recovery and viability of sperm in ICSI pipette after ultra rapid freezing or slow freezing. Fertil Steril. 2003; 80
- Tomita K, Sakai S, Khanmohammadi M, Yamochi T, Hashimoto S, Anzai M, et al. Cryopreservation of a small number of human sperm using enzymatically fabricated, hollow hyaluronan microcapsules handled by conventional ICSI procedures. J Assist Reprod Genet. 2016; 33(4):501-11.
- Organization WH. Geneva: WHO Laboratory Manual for the Examination and Processing of Human Semen Switzerland; 2010.
- Fakhrildin MBM, Flayyih N. A new simple medium for in vitro sperm activation of asthenozoospermic patients using direct swim-up technique. Kufa Med Journal. 2011; 14(1):67-75.
- Ledda S, Idda A, Kelly J, Ariu F, Bogliolo L, Bebbere D. A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor. J Assist Reprod Genet. 2016; 33(4):513-8.
- Just A, Gruber I, Wober M, Lahodny J, Obruca A, Strohmer H. Novel method for the cryopreservation of testicular sperm and ejaculated spermatozoa from patients with severe oligospermia: a pilot study. Fertil Steril. 2004; 82(2):445-7.
- Keros V, Rosenlund B, Hultenby K, Aghajanova L, Levkov L, Hovatta O. Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants. Hum Reprod. 2005; 20(6):1676-87.
- Sieme H, Oldenhof H, Wolkers WF. Mode of action of cryoprotectants for sperm preservation. Anim Reprod Sci. 2016; 169:2-5.
- Zhang X, Zhou Y, Xia W, Wu H, Yao K, Liu H, et al. Effect of pre-freezing conditions on the progressive motility recovery rate of human frozen spermatozoa. Andrologia. 2012; 44(5):343-8.
- Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci. 2000; 60-61:481-92.
- Le MT, Nguyen TTT, Nguyen TT, Nguyen VT, Nguyen TTA, Nguyen VQH, et al. Cryopreservation of human spermatozoa by vitrification versus conventional rapid freezing: Effects on motility, viability, morphology and cellular defects. Eur J Obstet Gynecol Reprod Biol. 2019; 234:14-20.
)
