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Cryopreservation of human spermatozoa: Comparison of TEST-yolk buffer and glycerol

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Abstract

Comparison of human sperm motility, morphology, and sperm membrane integrity using two cryoprotective media, Test-yolk buffer with glycerol and glycerol alone. Semen samples from 10 healthy donors were divided and frozen with either glycerol or a combination of glycerol and TEST-yolk buffer. Semen characteristics were evaluated before freezing and after thawing. Motility was measured at 0, 60, 120, and 180 minutes post-thaw following removal of the cryoprotectant (post-wash). Percentage of motile sperm decreased significantly compared to prefreeze values in both groups. Post-thaw motility following removal of the freezing media was higher in specimens that were frozen in TEST-yolk buffer compared to those frozen in glycerol at 0 minutes (P = 0.004). Similarly, specimens cryopreserved in TEST-yolk buffer had higher sperm motility compared to aliquots that were frozen in glycerol at 180 minutes (P = 0.013). The percentage of normal sperm forms was significantly higher post-wash and post-thaw in specimens that were cryopreserved in TEST-yolk buffer (P = 0.04). Sperm cryopreserved in TEST-yolk buffer had significantly better motility, morphology, and sperm membrane integrity than sperm preserved in glycerol alone.

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... However, the results were highly controversial in the literature. This debate could be due to differences in the study population characteristics, in the type of solutions compared together, and in the parameters analysed on raw semen or on washed spermatozoa (Hallak et al., 2000;Hammadeh et al., 2001, Nallella et al., 2004Abush et al., 2014;Fabozzi et al., 2016). ...
... In this report, we performed the cryopreservation on the raw unwashed semen, because it was reported that the presence of seminal plasma during the freezing process provides better outcome after thawing (Lannou, 1999;Donnelly et al., 2001;Fabozzi et al., 2016). Despite that, the seminal fluid does not provide a full protection, and sperm cryoinjury can still occur in good and bad semen quality (Lannou, 1999;Hallak et al., 2000;Hammadeh et al., 2001). This could explain the results of our study. ...
... To the best of our knowledge, this is the first study comparing TYB medium to Sydney IVF Sperm Cryopreservation Buffer (Cook Medical). In parallel, these findings are in accordance with several reports, showing that TYB medium has a superior protective power when compared to other media (Ragni et al., 1993;Hallak et al., 2000;Stanic et al., 2000;Fabozzi et al., 2016). Of particular interest, Nallella et al. (2004) showed that post-thaw motility and CSF were higher using TYB medium (Irvine Scientific) compared to the Sperm Freezing Medium (Medicult) and Enhance Sperm Freeze (Conception Technologies). ...
... The protocol requires the entire volume of freezing medium to be added at one time followed by immersion of specimens in liquid nitrogen (Nallella et al. 2004;Kobayashi et al. 2001b). Contrary to the idea that gradual acclimatization to very low temperatures protects sperm functional integrity to a greater extent, several studies have shown the IS method to be superior to slow freezing in terms of better post-thaw sperm motility and cryosurvival (Nallella et al. 2004;Hallak et al. 2000). ...
... Sperm cells are highly permeable to glycerol, which is a commonly used cryoprotectant (Hallak et al. 2000). Glycerol is often supplemented with citrate or egg yolk, which act as cryobuffers because they contain macromolecules that do not permeate the cell membranes of the sperm. ...
... In a study that compared TYB to proprietary media which are commonly used cryobuffer solutions such as Sperm Freezing Medium (Cooper Surgical, Trumbull, CT) and Enhance Sperm Freeze (Conception Technologies, San Diego, CA), the sperm that was cryopreserved in TYB was shown to have the longest longevity (Hallak et al. 2000). Sperm membranes may be stabilized by an altered phospholipid: cholesterol ratio imposed by the TYB, which may in turn reduce damage by free radicals. ...
Chapter
Cryopreservation is the collection, freezing, and long-term storage of sperm, and is a highly effective method of protecting male fertility potential. Cryopreservation of semen is a vital procedure which can be employed for a variety of purposes, including donor insemina-tion and the preservation of gametes in patients under-going gonadotoxic treatment. It also may be helpful to fertile couples who experience difficulty conceiving (Thomson et al. 2009). The increasing success of cancer treatment and concerted efforts to ensure quality of life after successful treatment have placed great emphasis on the need to preserve the reproductive capability of young men. Many health care professionals agree that the option to bank one's sperm should be offered systematically to all patients who may benefit, including those at risk for future infertility such as patients about to undergo cytotoxic chemotherapy (Achille et al. 2006; Kliesch et al. 1997a). However, this recommendation has yet to become standard practice. In a 2002 survey, only 10% of American physicians reported offering sperm Contents
... Semen analysis was done as recommended by WHO (1999) [8,9]. Sperm activation was done by using (SLM) and centrifugation method with a FertiCultTM medium [10,17]. Preparation of culture media and cryostorage of semen patients was made as described by Al-Dujaily and Al-Shammary [11] and Al-Taee [12]. ...
... Adding the egg-yolk buffer to the semen with a high viscosity can improve the percentage of sperm motility, viability and morphology. [17]. The data of this study revealed a highly significant improvement of all sperm parameters after 24 hr cryostorage of fertile samples when compared with infertile samples. ...
... Supplements can always be included in vitro to compensate for this deficiency. Multiple studies show the advantage of using TEST-yolk buffer as a cryoprotectant with glycerol to prevent damage in spermatozoa (Hallak et al., 2000;Nallella et al., 2004). Using TEST-yolk buffer in combination with glycerol is preferred compared with using glycerol alone. ...
... Using TEST-yolk buffer in combination with glycerol is preferred compared with using glycerol alone. This combined approach was shown to preserve higher motility, morphology and sperm membrane integrity (Hallak et al., 2000). It also decreases the number of spermatozoa with phosphatidylserine externalization (Duru et al., 2001) and prevents excess chromatin structure damage and morphology changes (Hammadeh et al., 2001) ...
Article
Apoptosis is an ongoing physiological phenomenon that has been documented to play a role in male infertility, if deregulated. Caspase activation, externalization of phosphatidylserine, alteration of mitochondrial membrane potential and DNA fragmentation are markers of apoptosis found in ejaculated human spermatozoa. These markers appear in excess in subfertile men and functionally incompetent spermatozoa. Sperm cryopreservation is a widely used procedure in the context of assisted reproductive techniques. Cryopreservation and thawing is a procedure that inflicts irreversible injury on human spermatozoa. The damage is manifested by a decrease in recovery of viable spermatozoa with optimum fertilization potential. This review describes the implication of apoptosis as one of the possible mechanisms involved in sperm cryoinjury. Evidence shows significant increase in some apoptosis markers following cryopreservation and thawing. On the other hand, the increase in sperm DNA fragmentation following cryopreservation and thawing requires further investigation. Specific technical measures should be applied to minimize the induction of apoptosis in human spermatozoa during cryopreservation and thawing. These include standardization of freezing protocols and cryoprotectant use. Selection of non-apoptotic spermatozoa may also prove to be of benefit.
... Moreover, the addition of amides and saccharides such as sucrose is essential to prevent the swelling of cells induced by osmotic pressure, as well as the edema induced by low-Mw CPAs during thawing [12,17]. Therefore, non-permeating macro-Mw CPAs, such as test yolk buffer (TYB) [18,19] and human serum albumin (HSA) [20], should be added to the cryopreservation medium to prevent intracellular ice crystal formation induced by extracellular ice crystal formation. ...
Article
Full-text available
In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.
... The need for effective sperm preparation methods has increased with the increased use of assisted reproductive techniques. Sperm preparation techniques vary greatly in terms of recovery rates, motility, morphology, and degree of DNA damage [10, 14, 15]. Due to their simplicity, reproducibility, and excellent yields in motile spermatozoa, Percoll gradients became very popular for processing semen specimens. ...
Article
To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10(6) vs. 17.6 x 10(6)), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART.
... Cryosurvival of human sperm depends mainly on the cryoprotectant, the freezing technique and the initial quality of the specimen. The use of glycerol to prevent injury to human spermatozoa during cryopreservation is well established (18), and its association with buffers, such as Tris (hydroximethyl amino metano) and TES (n-Tris [hydroximethyl] methyl-2-amino-ethane sulphonic acid), and eggyolk yield optimal cryosurvival rates (19). Slow freezing using programmable freezing machines or fast freezing, as used in this study, seems to have no direct effect on thaw survival both in normal and poor quality sperm (20). ...
Article
Full-text available
To study the resistance of human spermatozoa to cryoinjury in repeated cycles of thaw-refreezing by using the fast liquid nitrogen vapor method. Semen specimens were obtained from sixteen normal and oligozoospermic individuals who required disposal at the sperm bank. Five of them had testicular cancer. Specimens were thawed and an aliquot was removed for analysis. The remaining specimens were refrozen without removing the cryomedia. Repeated freeze-thaw cycles were performed until no motile sperm were observed. Sperm motility, number of motile spermatozoa and viability were determined after thawing. Resistance to cryoinjury was compared between groups and also after each refreezing cycle within groups. Motile spermatozoa were recovered after five and two refreeze-thawing cycles in normozoospermic and oligozoospermic specimens, respectively. There were no significant differences in the recovery of motile spermatozoa between thaws within each group of normal and oligozoospermic specimens, but percentage motility and total number of motile spermatozoa were significantly lower in the oligozoospermic one. Specimens from men with cancer were exposed to six refreeze-thawing cycles. Although recovery of motile spermatozoa was significantly impaired after each thawing, there were no significant differences in the recovery of motile sperm between thaws in cancer and non-cancer groups. Human spermatozoa resist repeated cryopreservation using the fast liquid nitrogen vapor method. Normozoospermic specimens withstand refreezing for an average two cycles longer than oligozoospermic ones. Specimens from cancer patients seem to resist repeated cryoinjury similarly to non-cancer counterparts. Resistance to repeated cryoinjury was related to the initial semen quality.
... In an attempt to reduce chilling injury and improve the optimal survival and fertility capacity of human sperm following cryopreservation, many different cryoprotectant media have been developed Currently, the most widely used cryoprotectant is the permeating agent glycerol, as it has been confirmed the most effective in lowering the freezing point of intracellular water (Royere et al., 1996). Other compounds are added to glycerol-containing media as buffers to yield optimal crysurvival rates (Hallak et al., 2000), In spite of these advances, a gold-standard method of cryopreserving human semen with a optimal cryoprotectant is yet to be determined. ...
Chapter
Full-text available
Here, an overview of the various clinical uses of processing of semen samples and cryopreservation is given. The effects of sperm processing on conventional semen parameters are discussed along with the ramifications of removing seminal plasma, oxidative stress and potential benefits of antioxidant addition in laboratory processing of testicular and ejaculated sperm. In the second part of the chapter, the effects of a second laboratory hazard, namely, cryopreservation, are discussed in terms of effects on conventional parameters of sperm structure and function and also on DNA fragmentation of testicular and ejaculated sperm. The greater vulnerability of sperm from infertile men is described, as well as the cryoinjury displayed by those with teratozoospermia. The mechanisms of cryoinjury are set out with special reference to oxidative damage and the process of repeated freezing and thawing. The efficacy of commercially available cryoprotectants is also discussed. Finally, novel freezing–thawing protocols such as freeze-drying and vitrification of sperm are explored. KeywordsSperm processing-Cryopreservation of sperm DNA-Sperm DNA integrity-Sperm DNA processing-Density-gradient centrifugation
... Twenty-four hours after the semen was frozen, a vial was removed and thawed by incubation at 37C for 20 minutes. A 5-L aliquot was analyzed as described above (Hallak et al, 2000). ...
Article
Cytotoxic drugs and immunosuppressive therapies are used to treat patients with nonmalignant, nontesticular systemic diseases. These therapies can permanently suppress spermatogenesis. Sperm cryopreservation before treatment theoretically could give these men the opportunity to achieve a pregnancy with a woman later in life when the couple decides to do so. However, it is not known whether pretreatment sperm quality in these men is good enough to be used for assisted reproductive techniques. The main objective of this study was to determine the usefulness of cryopreservation in this patient population by: 1) assessing their pretreatment semen quality (eg, count, motility, and motion kinetics) and comparing it with that of healthy donors before and after cryopreservation; 2) comparing patients' pretreatment semen characteristics with World Health Organization reference values for normal sperm; and 3) examining the differences in semen parameters among patient groups. Semen specimens were obtained from 25 healthy donors and from 23 patients with a variety of disorders (12 had autoimmune disorders, 4 had kidney disorders, 3 had diabetes, 2 had ulcerative colitis, and 2 had heart transplants). All patients, except those with diabetes, required immunosuppressive or cytotoxic therapy. Although the pretreatment quality of the semen of these patients was not as good as that of donors, semen samples were within the normal reference range of the World Health Organization. No statistically significant differences in sperm parameters were found within the 4 patient groups except for those with diabetes (n = 3), who showed poorer sperm counts (P < .04). However, no conclusive evidence can be reached due to the small sample size. Our results indicate that pretreatment semen quality in these patients is adequate for reproductive techniques. We believe that cryopreservation should be offered to patients of reproductive age with disease or treatment regimens that may cause infertility.
... Damage from freezing can be minimized by optimizing freezing rates and using cryopreservatives. To optimize post-freezing motility, several different cryopreservation methods have been tested to determine optimal freezing rates and cryopreservative (Verheyen et al., 1993;Morris et al., 1999;Gilmore et al., 2000;Hallak et al., 2000). ...
Article
Full-text available
Although the comet assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the comet assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. The highest correlation was between comet assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral comet assay.
... The CASA setting was as follows: frame rate (frames/second): 30; duration of data capture (frames): 15; minimum motile speed (lm/s): 600; distance scale factor (lm/pixel): 0.9457; centroid cell size minimum (pixels): 2; centroid cell size maximum (pixels): 8; number of cells to find per well: 200; and the minimum number of fields per sample: 3. All semen specimens were cryopreserved using Test-yolk buffer (TYB, Irvine Scientific, Santa Ana, CA), a glycerol-based cryoprotectant [7]. An aliquot of the freezing medium equal to 25% of the semen sample volume was added to the specimen and gently mixed for 5 min using a Hema-Tek aliquot mixer (Miles Scientific, Elkhart, IN). ...
Article
Semen cryopreservation is a useful tool for preserving fertility in men who have been diagnosed with cancer and will undergo chemotherapy, radiotherapy or testicular surgery. Semen is also commonly cryopreserved prior to its use in assisted reproductive techniques such as in vitro fertilization and intracytoplasmic sperm injection. The post-thaw quality of banked sperm can vary, which may negatively affect fertilization rates. The objective of our study was to assess the pre-freeze and post-thaw variability of sperm parameters in patients who used our sperm banking services. Multiple samples obtained after a short period of sexual abstinence were examined for variation in sperm characteristics. Semen samples showed a high degree of post-thaw inter-sample variability in sperm motility, motion characteristics, and percentage cryosurvival rate compared with the pre-freeze inter-sample variability. Further research is necessary to understand the mechanism(s) responsible for this variability. This may also assist clinicians utilize semen samples with optimum semen quality in ART procedures.
... Furthermore, researchers at Cleveland Clinic compared TYB with other commer cially available media, such as Sperm Freezing Medium (Cooper Surgical, Trumbull, CT) and Enhance Sperm Freeze (Conception Technologies, San Diego, CA), and found that the sperm cryopreserved in TYB had the greatest longevity. 75 Three possibilities may explain such findings: (a) sperm membranes may be stabilized by an altered phospholipidcholesterol ratio imposed by the egg yolk content of TYB, (b) egg yolk has free radical scavenging properties resulting in less peroxi dative damage to the sperm and, (c) TYB has a nearly optimal concentration of glycerol. Because sperm have critical volume limits, CPM should be added slowly and in a stepwise fashion with shaker assistance to reduce the risk of osmotic stress (Figure 7). ...
... Egg yolk, on the other hand, which is not a cryoprotectant itself and is often used in combination with glycerol, seems to confer improved sperm plasma membrane fluidity, resulting in improved cryo-survival. 49 Prins et al. compared eight different cryopreservatives and concluded that sperm frozen with egg yolk buffer demonstrated the highest post-thaw survival. 50 Mahadevan and Trounson have developed a modified Tyrod's medium containing 7.5 percent glycerol, referred to as Human Sperm Preservation Medium (HSPM). ...
Chapter
banking with cryopreservation is an important proce-dure that can be used to preserve the future fertility of men who are facing the prospect of permanent loss of fertility for several reasons. For example, some patients who choose sperm banking have been diagnosed with cancer and are about to undergo gonadal surgery or gonadotoxic treatment such as chemotherapy and/or radiation therapy. In other cases, men are already infer-tile due to azoospermia or sexual dysfunction but have some viable sperm that can be successfully harvested and frozen. In any case, when pregnancy is desired, the sperm sample can be thawed and used in a number of assisted reproductive tech-niques (ARTs): intracytoplasmic sperm injection (ICSI), intra-uterine insemination (IUI) and in vitro fertilization (IVF). This chapter will discuss the current role of sperm banking with cryo-preservation, including the main indications, procedures used to extract, process and freeze sperm, and ART outcomes. We will also discuss the need to educate patients about sperm banking before treatment. Absent Partner When either the male or female partner is often absent (due to travelling, for example) it can be difficult to time intercourse with ovulation. About 12 percent of couples are unable to conceive after one year of unprotected intercourse and are therefore considered infer-tile. 1 About 30–40 percent of these couples are unable to conceive due to male factor infertility. Approximately 10 percent of male factor infertility is caused by azoospermia. In the most severe cases of male infertility, couples may decide to use cryopreserved sperm from a healthy third-party donor. 2
... A final concentration of 7.5% has been shown to be an optimal concentration of glycerol for freezing solution. Egg yolk on the other hand, which is not a cryoprectant itself and often used in combination with glycerol, seems to confer improved sperm plasma membrane fluidity, resulting in improvement in cryo-survival (Hallak et al. 2000). ...
Article
Full-text available
Sperm cryopreservation is an important part of an infertility program for patients undergoing infertility treatments, fertility assurance for vasectomy cases, and for fertility preservation due to cancer or other medical conditions. With recent developments in reproductive technology, even men with severely impaired sperm parameters can benefit from cryopreservation as procedures such as intra-cytoplasmic sperm injection (ICSI) require only a few sperm to achieve fertilization and pregnancy. The increasing success of cancer treatment and concerted efforts to ensure quality of life after successful treatment have placed great emphasis on the need to preserve the reproductive capability of young men. It is a highly effective method of protecting male fertility potential, and involves collection, freezing, and long-term storage of sperm. Based on the etiological condition of the patients, sperm can be collected by ejaculation or by surgical retrieval from epididymis or testes. The option to bank sperm should be offered systematically to all patients who may benefit. However, this is not a standard of practice yet; it may be overlooked due to lack of physician awareness regarding the need for fertility preservation and the effectiveness of this option, and/or overestimating the limitations of poor baseline sperm quality leading physician to view cryopreservation as futile. Failure to offer cryopreservation ignores the only possible reproductive option available to certain patients.
... The results of these in vitro sperm function tests are encouraging, suggesting that it is feasible to replace hen's egg yolk with soy lecithin, at least with regard to in vitro sperm function assays, as it is possible that cryopreservation and thawing could effect subtle alterations in the sperm cells not identifiable by evaluation of motility alone (20). Cryoprotectant medium that has glycerol but no egg yolk is capable of providing viable human sperm after thawing; however, egg yolk has been found to support superior postthaw outcomes compared with glycerol alone (21)(22)(23)(24). Jeyendran et al. (8) demonstrated that postthaw motility was superior after cryopreservation in medium with egg yolk plus glycerol compared with medium with soy lecithin and glycerol; however, replacement of 2% v/v glycerol with 2% v/v DMSO improved postthaw motility, similar to egg yolk plus glycerol. ...
Article
Full-text available
Semen specimens (one ejaculate from each of 20 consenting study participants) were subjected to routine semen analysis, an in vitro sperm binding assay (HBA), and a sperm chromatin dispersion assay (HaloSperm), both before and after cryopreservation using cryoprotectant media supplemented with either egg yolk or soy lecithin. Comparing the equivalency of the two phospholipid cryopreservation supplements with regard to postthaw functional parameters demonstrated that there were no statistically significant differences between the two supplements for [1] recovery of motile sperm, [2] maintenance of sperm cell morphology, [3] maintenance of the ability of sperm to bind to hyaluronate in vitro, or [4] maintenance of sperm DNA integrity.
... The methodological characteristics of the included studies including study design, number of participants and procedure-specific characteristics were collected. We developed a quality assessment form using the validity criteria proposed by Juni et al. for clinical trials and the Newcastle– Ottawa Scale for cohort studies (Hallak et al., 2000; Juni et al., 2001; Wells et al., 2007). Quality evaluation was performed by two reviewers independently (M.B., F.A.) and any disagreement was resolved by consensus. ...
Article
Full-text available
Despite interest in cryopreservation of individual or small number of human spermatozoa, to date, little data is available as regards its effectiveness. We systematically reviewed the outcome after cryopreservation of individual or small numbers of human spermatozoa in patients with severe male factor of infertility. We searched the MEDLINE, EMBASE, Cochrane Systematic Reviews, CENTRAL, Web of Science, Scopus databases for relevant studies up to June of 2008. The search used terms referring to cryopreservation of small amount of sperm. Included studies were limited to human studies with no language restrictions. We identified 30 reports including 9 carriers used for cryopreservation of small quantities/numbers of human spermatozoa (7 non-biological and 2 biological carriers). A wide variety of cryopreservation vehicles were reported. The recovery rate of spermatozoa cryopreserved in a known small number varied widely from 59 to 100%. Fertilization rates were in the range of 18-67%. Frozen-thawed spermatozoa, using this method, were subsequently used for intracytoplasmic sperm injection in only five studies, with few pregnancies reported so far. To date, there remains no consensus as to the ideal carrier for cryopreservation of small number of spermatozoa for clinical purposes. Cryopreservation of individual or small numbers of human spermatozoa may replace the need for repeated surgical sperm retrieval. A controlled multicenter trial with sufficient follow-up would provide valid evidence of the potential benefit of this approach.
... Glycerol is a commonly used cryoprotectant [ 49 ]. It is often supplemented with citrate or egg yolk, which acts as cryobuffer because they contain macromolecules that do not permeate the cell membranes of the sperm. ...
Chapter
As cancer survival rates increase worldwide, sperm banking is gaining popularity as a way to preserve male fertility. Banked sperm can be used in assisted reproductive procedures to initiate a successful pregnancy, thereby increasing the likelihood of male cancer patients fathering biological children. The American Society of Clinical Oncology (ASCO) advocates sperm cryopreservation as an effective method of fertility preservation in young men with cancer. However, the number of cancer patients utilizing sperm banking options remains low for a variety of reasons. In this chapter, we describe the incidence of cancer among adults and adolescents seeking fertility preservation before treatment, sperm banking techniques, and the challenges that limit the use of this technology among cancer survivors.
... The semen samples were cryopreserved with freezing medium TEST yolk buffer, consisting of TES (N-tris[hydroxymethyl]methyl-2-aminoethanesufonic acid), Tris (Tris[hydroxymethyl]aninomethane) and chicken oocyte yolk (TYB, 20% oocyte yolk and 12% glycerol, Irvine Scientific, Santa Ana, CA, USA, Catalogue No 9971), conditions known to result in the best vitality parameters (motility, morphology and membrane integrity) of spermatozoa after cryopreservation, as demonstrated previously (Glander and Schaller, 1999;Hallak et al., 2000). For cryopreservation, the semen samples were diluted drop-wise with an equivalent volume of TYB containing 12% (v/v) glycerol. ...
Article
Full-text available
Magnetic-activated cell sorting (MACS) using annexin V-conjugated microbeads in a liquid phase eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine (EPS) residues. The procedure allows the enrichment of a sperm population free of apoptosis markers, giving higher fertilization potential. Our aim was to determine if the annexin V binding principle can be transferred onto a glass wool filter system in order to produce a solid phase filter. Semen samples (n = 42) were subjected to a molecular glass wool filter system using glass surfaces coated with annexin V and compared with aliquots separated by conventional glass wool, as well as with annexin V-MACS. The extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labelled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and EPS using a fluorescein isothiocyanate-coupled monoclonal antibody. Annexin V-negative sperm filtered out by the newly developed molecular glass wool filtration (GWF) system displayed superior quality in terms of high MMP integrity, as well as, to a small extent, caspase 3 activation and EPS. The effect of traditional GWF can be further improved by combination with annexin V binding. This newly developed solid phase molecular filter system has been proven to enrich spermatozoa free of apoptosis markers to the same extent as the annexin V magnetic separation technique. The selection of spermatozoa free of apoptosis markers by molecular glass wool filters may enhance the results of IVF.
... This study evaluated the effect of MEL and CAF addition on the functional characteristics of normozoospermic sperm samples before cryopreservation and postthawing, using the slow freezing method and glycerol as a cryoprotectant. As expected, the cryopreservation process has had its panel of associated adverse effects reducing sperm motility and mitochondrial activity while increasing sperm ROS levels and DNA fragmentation [15,17,27,39,[62][63][64][65][66][67][68]. In the present study, sperm motility went down from 50.22% in fresh semen to 7.5% postthawing. ...
Article
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Cryopreservation processes can damage spermatozoa and impair structural and functional cell characteristics. Plasma, nuclear membranes, and cellular organelles can suffer from the freeze and thaw process. This study evaluates the protective and stimulant effect of melatonin and caffeine supplementation on the functional characteristics of human spermatozoa before and after freezing. Thirty seminal samples from normozoospermic men aged 19–45 years old collected between October 2012 and May 2017 were included. Semen samples were supplemented with either 2 mM melatonin (MEL) prior to cryopreservation, 2 mM caffeine (CAF) in postthaw, or CAF and MEL (CM) in precryopreservation and postthaw, respectively. Kinetics and seminal parameters, mitochondrial activity, DNA fragmentation, and reactive oxygen species (ROS) levels were analyzed before and after cryopreservation. A significant reduction in sperm concentration, total and progressive motility, sperm kinetics, and mitochondrial activity, as well as a significant increase in DNA fragmentation and ROS production in postthaw samples compared to fresh samples, was identified. After administration of a caffeine and/or melatonin supplement, there was a significant increase in progressive motility in the CAF ( p=0.005 ) and CM ( p=0.048 ) groups, as well as mitochondrial activity in the CM group ( p<0.05 ). Cryopreservation has negative effects on overall sperm quality and increases ROS production. A combination of caffeine and melatonin in prefreeze and postthaw sperm samples has proven to be a very effective and simple way to improve semen quality. This will be particularly useful for initial low-quality semen samples, those which suffer the most from the freezing/thawing process.
... During freezing-thawing process, sperm may deteriorate because of damaged cell membranes, impairing sperm motility, and altering its morphology (6) . Many factors affect the sperm quality in freezing and thawing e.g. the freezing method (7) , temperature control (8) , sperm preparation technique (9) , and type of cryopreservative agent (10) . ...
Article
Background: Cryoprotectant has a pivotal role in preventing cell injury during sperm cryopreservation. Objective: To compare the post-thawed sperm parameters between the use of glycerol egg yolk citrate (GEYC) and Sperm Freeze medium as cryoprotectant in human sperm cryopreservation. were included in the study. All of the semen samples were analysed with standard technique using the World Health Organization protocol. The semen samples were divided into 2 groups and mixed with GEYC and SpermFreeze medium respectively. Pre-freezing and post-thawed sperm parameters were analyzed with Computer-Assisted Sperm Analysis system. Results: A total of 30 volunteers who could collect their semen samples participated in the study. The mean and standard deviation of sperm concentration was 53.3+27.5 million/milliliter, the percentage of sperm motility was 68.2+22.1 and the percentage of normal sperm morphology was 4.7+1.6. When compared the semen samples that freezing them with GEYC or SpermFreeze medium, there was no significant difference in mean post-thawed sperm concentration (33.7+17.4 vs. 32.7+15.6 million/milliliter, p-value = 0.827) and percentage of sperm motility (38.6+25.6 vs. 35.1+28.1, p-value = 0.616). However, it had no significant effect on normal sperm morphology before and after freezing-thawing cycle and also, there was no significant difference in percentage of post-thawed normal sperm morphology between two cryomediums (4.4+1.3 vs. 4.4+1.4, p-value = 0.849). Conclusion: For normal semen samples, use of GEYC medium is an acceptable option in human sperm cryopreservation.
... On the other hand, repeated surgical procedures for diagnostic or therapeutic extraction will destroy the blood-testicular barrier and impair sexual function. Recently, several cryopreservation methods have been reported, and various containers and novel methodologies have been established to preserve the small number of spermatozoa [20][21][22][23][24][25][26][27][28], Successful pregnancies have been reported through ICSI, with a few spermatozoa cryopreserved in empty zona pellucida [29]. Most recently, Endo Y et al. reported successful delivery after transfer of a blastocyst derived from ICSI using limited numbers of sperm stored in a cell sleeper [30], despite the fact that they did not obtain motile spermatozoa after warming them from the cell sleeper and cryotop containers. ...
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PurposePatients with Klinefelter syndrome (KS) who receive assisted reproductive technology (ART) treatment often experience poor pregnancy rates due to decreased fertilization, cleavage, and implantation rates and even an increased miscarriage rate. Mounting evidence from recent studies has shown that various technological advances and approaches could facilitate the success of ART treatment for KS patients. In this review, we summarize the methods for guiding KS patients during ART and for developing optimal strategies for preserving fertility, improving pregnancy rate and live birth rate, and avoiding the birth of KS infants.Methods We searched PubMed and Google Scholar publications related to KS patients on topics of controlled ovarian stimulation protocols, sperm extraction, fertility preservation, gamete artificial activation, round spermatid injection (ROSI), and non-invasive prenatal screening (PGD) methods.ResultsThis review outlines the different ovulation-inducing treatments for female partners according to the individual sperm status in the KS patient. We further summarize the methods of retrieving sperm, storing, and freezing rare sperm. We reviewed different methods of gamete artificial activation and discussed the feasibility of ROSI for sterile KS patients who absolutely lack sperm. The activation of eggs in the process of intracytoplasmic sperm injection and non-invasive PGD are urgently needed to prevent the birth of KS infants.Conclusion The integrated strategies will pave the way for the establishment of ART treatment approaches and improve the clinical outcome for KS patients.
... We started to study the genetic causes of male infertility by initially mapping the Y chromosome in azoospermic men and fertile male control populations, eventually leading to the complete sequencing of the Y chromosome, almost in parallel with the production of intracytoplasmic sperm injection (ICSi) and testicular sperm extraction (TESE) for the purposes of azoospermia in 1993. In the end, this contributed to a clarification as to why the testis of azoospermic men, generally expected to produce no sperm, still contain small quantities of it [10][11][12][13][14][15][16]. Subsequently, much of the work on male infertility genetics focused on Y chromosome aberrations. ...
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The aim of the present study was to evaluate the effect of different vitrification protocols on reactive oxygen species (ROS) and apoptosis in human ovarian tissue. Human ovarian tissue pieces were exposed to different vitrification solutions. The intracellular redox state level was measured using the fluorescent dye dichlorodihydrofluorescein diacetate. Imaging of apoptotic cells was monitored by anti-caspase-3 immunolabelling after vitrification and warming. Following equilibration in either 40% ethylene glycol (EG) (v/v), 0.35 M sucrose + 10% egg yolk extract (v/v) or 40% EG (v/v), 18% Ficoll-70 (w/v) + 0.35 M sucrose for 6 min, ovarian pieces were cooled to -196 degrees C using four different protocols. Tissue that was cooled very rapidly (plunged directly into liquid nitrogen in straws or on grids or plunged directly into metal filings precooled to -196 degrees C) showed no statistically significant increase in either tissue ROS levels or the number of apoptotic cells after warming. In contrast, cooling using a less rapid method (nitrogen vapour at -120 degrees C) resulted in significantly elevated ROS levels and apoptosis after warming. There were no significant differences between the two vitrification solutions. This indicates that human ovarian tissue pieces should be vitrified using very rapid cooling rates.
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Freezing of human sperm is considered a routine procedure in assisted reproductive technology (ART) laboratories. This article considers various aspects of cryopreservation of human sperm. Human sperm show a specific cryophysical behaviour and different sperm freezing protocols have been developed to avoid damage to the sperm cells. The damage can range from impaired motility and reduced viability to damage to the cellular organelles and effects at the molecular level, resulting in an impaired fertilizing potential. As testicular sperm are immature and only a small number can be retrieved, special techniques are required for successful freezing and thawing of these samples. Banking of human sperm has to be performed in a safe and controlled way and different guidelines are necessary to ensure that this is achieved.
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Studies were carried out to analyze the cryoprotecting efficacy of several amino acids by use of a chemically defined synthetic medium (modified Ringer's solution) and goat cauda epididymal sperm as the model system. Motile goat cauda sperm dispersed in the synthetic medium were subjected to a freezing protocol in a computer-controlled bio-freezer, cooling 0.25°C min-1 to 5°C, 5°C min-1 to -20°C, and 20°C min-1 to -100°C, prior to being plunged into liquid nitrogen. In the absence of amino acids, sperm cells completely lost their flagellar motility. Of all the amino acids tested, L-alanine showed maximal cryoprotection potential. L-Alanine at 135 mM offered optimum cryoprotection potential: recovery of sperm forward motility and total motility were 14 ± 2% and 19 ± 2%, respectively. L-Glutamine, L-proline, and glycine at optimum concentration (100-150 mM) cryopreserved approx. 11-17% total motility of the sperm cells, whereas amino acids such as L-arginine, L-lysine, and L-histidine offered little cryoprotection (0-5%) to the cells. Increasing the amino acid concentration beyond the optimum level sharply decreased the recovery of the sperm motility, which therefore showed a biphasic cryoprotection profile. Addition of amino acids enhanced (approx. 7-10%) the cryoprotection efficacy of the well-known cryoprotectants glycerol and a combination of glycerol and dimethyl sulfoxide. The presence of glycerol caused a marked reduction (from 100-150 mM to 20-70 mM levels) in the optimal cryoprotective concentration of the amino acids. The combined cryoprotecting action of glycerol, dimethyl sulfoxide, and amino acids provided motility recovery as high as 52%. The observation that amino acids and dimethyl sulfoxide had an additive effect in augmenting the cryoprotecting potential of glycerol suggests that the mechanism of their action is different from that of glycerol. This cocktail of cryoprotectants may be useful for cryopreservation of semen of various species.
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To evaluate the effect of magnetic-activated cell sorting in cryopreservation-thawing protocols on sperm motility and cryosurvival rate. Prospective-controlled study. Andrology department at a university-based medical institution. Ten healthy volunteer sperm donors. Sperm populations were separated using annexin-V magnetic-activated cell sorting before and after the cryopreservation-thawing process. Sperm motility and cryosurvival rate. Annexin-negative sperm separated by magnetic-activated cell sorting had statistically significantly higher motility following cryopreservation-thawing than sperm that were not separated. Similarly, annexin-negative spermatozoa also had higher cryosurvival rate than sperm cryopreserved without magnetic-activated cell sorting and sperm that were annexin-positive. Superparamagnetic annexin V-conjugated microbeads can separate spermatozoa with externalized phosphatidylserine, which is considered one of the early features of late apoptosis. The separation of a distinctive population of nonapoptotic spermatozoa with intact membranes may optimize the cryopreservation-thawing outcome. Magnetic-activated cell sorting using annexin-V microbeads enhances sperm motility and cryosurvival rates following cryopreservation.
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As a new method for preservating human spermatozoa, freeze drying has many special advantages. However, the freeze drying process could damage the the DNA of the human spermatozoa, the injury will also be caused by the endoenzyme of the spermatozoa. So far, the commonly used freeze drying protective agents-ETBS (ethylenedioxy-diethylene- dinitrilo-tetraacetic acid (EDTA) or ethylene glycol-bis-(2- aminoethyl)-N,N,N', N'-tetraacetic acid (EGTA) was added to the Tris-HCl buffer) has been proved to restrain the enzyme activation in sperm and protect the genetic material. Many studies showed that yolk and trehalose can supply special protection for the sperm in the freeze drying process. In this study, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and single cell gel electrophoresis (SCGE) were used to detect the sperm DNA damage after freeze drying with ETBS, ETBS added with trehalose or added with both trehalose and yolk as protective agents.
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OBJETIVOS: avaliar as características do sêmen humano preservado a +4ºC e a -196ºC por 24 h e determinar a técnica ideal para utilização em procedimentos específicos. MÉTODOS: amostras de sêmen de 24 voluntários foram analisadas após a coleta e divididas em duas alíquotas, uma resfriada em a +4ºC e outra congelada a -196ºC. As amostras foram mantidas em baixas temperaturas por 24 h e então em temperatura ambiente por 30 minutos (T1), capacitadas (T2) e incubadas a +37ºC por 90 minutos (T3), sendo avaliadas quanto à concentração e motilidade progressiva em T1, T2 e T3. Para análise dos resultados obtidos com as duas diferentes técnicas foi utilizado o Modelo Linear Geral e para análise dos dados obtidos com a mesma técnica, em dois diferentes momentos de observação, foi utilizado o teste de Wilcoxon (a de 5% e p0,05) quanto à concentração, motilidade e NTEM entre as técnicas nos três momentos de observação. Tampouco houve diferença entre as variáveis após capacitação e após incubação no sêmen resfriado, mas, no congelado, a concentração foi significativamente superior após capacitação. CONCLUSÕES: embora a concentração e a motilidade progressiva não tenham diferido em ambas as técnicas, sugere-se o uso do resfriamento em procedimentos específicos a curto prazo, devido à simplicidade e baixo custo. Quando o sêmen congelado for necessário, recomenda-se a utilização logo após a capacitação para evitar redução da qualidade do mesmo.
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This study examined the effects of cryopreservation on DNA integrity of spermatozoa from 34 fertile subjects and 166 infertile subjects comprised of 80 teratospermic, 32 normospermic, 30 astheno-teratospermic, and 24 oligo-astheno-teratospermic individuals. Semen samples were prepared by swim-up and the Percoll density gradient centrifugation method (Pdgc) prior to freezing in liquid nitrogen. Neat and prepared samples were supplemented with cryoprotectant (SpermFreez) in cryoampoules and were frozen using the static phase vapor cooling procedure. Sperm DNA integrity of all thawed samples was determined using the alkaline comet assay. It was noticed that the sperm DNA integrity of frozen samples of fertile subjects was considerably higher than that of infertile subjects with greater catch-up integrity similar to the fresh samples. Freezing caused less chromatin damage to sperm of Pdgc samples from both fertile and infertile subjects as was compared to the neat and swim-up samples. It is concluded that the increase in comet frequency of frozen-thawed samples from infertile subjects was more prominent (8.25-22.78%; P<0.01) than in the fresh samples. Frozen-thawed samples from Ts (Teratospermic individuals) and ATs (Astheno-teratozoosspermic) showed higher level of OTM (Olive tail moment) indicating a higher level of chromatin fragmentation than fertile, Ns (Normospermics), and OATs (Oligo-astheno-teratozoospermics).
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Cryopreservation of semen is a common procedure, both for autologous sperm banking and for donor sperm insemination. Additionally, new techniques employing semen banking are now common, including the use of epididymal aspirates and testicular biopsy specimens used for assisted reproductive therapies (ART). This chapter explores the regulatory, scientific, and practical aspects of sperm cryopreservation and banking.
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PURPOSE: to compare the characteristics of human semen preserved at +4oC and at –196oC for 24 h and to determine which technique is indicated for use in specific procedures. METHODS: semen samples of 24 voluntaries were analyzed after collection and divided into two aliquots, one of them cooled (+4oC) and the other frozen (–196oC). Samples were kept at low temperatures for 24 h and then at room temperature for 30 minutes (T1), capacitated (T2) and kept at +37oC for 90 minutes (T3), being analyzed regarding count and progressive motility at T1, T2 and T3. The General Linear Model was used to analyze results obtained with different techniques, while Wilcoxon's test was used to compare results obtained in two different moments using the same technique (a = 5% e p<0,05). RESULTS: data were missed in one fresh semen sample, in one sample after preservation, in five samples after capacitation and in two samples after incubation. The average number of total motile sperm/mL (NTMS) in fresh semen was 39.7 million (1.3-104.0). After preservation, the average NTMS in cooled semen was 9.6 million (0-37.4) and in frozen semen 8.7 million (0-41.2). After capacitation, the average NTMS was 5.4 million either in cooled (0-21.7), or in frozen semen (0-28). After incubation, the average NTMS in cooled semen was 9.8 million (0-40.5) and in frozen 4.4 million (0-25.6). Concerning count, progressive motility and NTMS, there was no significant difference (p>0,05) between techniques in the three moments of observation. In cooled samples, there was no difference between variables after capacitation and after incubation, but, in frozen semen, count was significantly greater after capacitation. CONCLUSIONS: although there has been no significant difference between semen count and progressive motility in both techniques, the use of cooled semen is recommended for specific procedures within a short time period due to its simplicity and low cost. When frozen semen is necessary, we recommend its use soon after capacitation in order to avoid loss in quality.
Article
The injury to the freeze-dried human spermatozoa is not only caused by the influence of preservation media to the physiological function, but also by the freezing and drying process. So far the selection of freeze-drying protective agents of sperm has focused on the view of physiological chemistry, aiming at restraining the enzyme activation in sperm. But the injury from freezing and drying was omitted. Many studies have showed that egg yolk could alleviate the freezing injury, and disaccharides (especially for trehalose) could offer special protection to cells during drying. In this work, trehalose was added to the freeze-drying protective agents that were commonly used by others, or trehalose and egg yolk were added together. According to the experimental results of Eosin Y staining, hypoosmotic swelling test and observation by microscopy, the protective functions of these media to the structure and membrane of the sperm were compared. The results implied that the media modified by trehalose could improve the protection to sperm, and the media supplemented with trehalose and egg yolk was the best.
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Human spermatozoa cryopreservation techniques are used to maintain and protect male fertility in cases such as infertility and malignancy treatments. However, during cryopreservation, the spermatozoa's metabolic rate is reduced and they undergo dramatic functional and structural changes owing to exposure to cryoprotectants and freezing-thawing procedures. While the effects of cryopreservation on cells are documented, to date the induced cryodamage on structural and/or functional sperm biomarkers is not well established at multivariate scale. To address this question, we performed basic sperm analysis, sperm DNA fragmentation assessment, spontaneous acrosome reaction measurement, and cytoskeleton evaluation after thawing samples from subjects with normal and low-quality semen. A cryodamage rate was used to determine the effects of the freeze-thaw process on spermatozoa. In addition, a Principal Component Analysis (PCA) was used for data reduction and to evaluate sperm-specific patterns during the cryopreservation process. We found that the vitality, progressive motility and sperm count from low-quality samples after cryopreservation show higher damage rates (≥40%) than in normal sperm samples. However, cytoskeleton, DNA, tail and mid-piece and acrosome display the highest cryodamage rates (∼50–99%) and are equally susceptible to cryopreservation-induced damage in both low- and normal-quality semen samples. Overall, the evaluation of these parameters provides meaningful information about different aspects of sperm functionality after cryopreservation.
Chapter
The number of cancer patients in young age has increased in the recent years, which is coupled with late marriages and family planning. This makes an important place for a new and emerging field of cryo-preservation of gametes for cancer patients. A significant proportion of oncological treatments utilize chemotherapy or radiation exposure, both of which are detrimental to spermatogenesis. In post-cancer treatment period, fertility may resume in some but not all patients. Therefore, these patients need to be counselled about the methods of fertility preservation before commencing anti-cancer therapy. The present chapter brings a comprehensive overview of the fertility preservation options for cancer patients and the techniques used in this process.
Chapter
Sperm cryopreservation utilizes state-of-the-art technology for fertility preservation in men who have diverse conditions that require freezing sperm for future use in ART. Sperm cryopreservation is of significance in order for these men with disease conditions to have a good quality of life. In this chapter we discuss the different indications for cryopreservation of sperm in men with cancer, infertility issues, extreme medical conditions and even posthumous sperm retrieval. The two major groups for sperm freezing, the auto-conservation group and the donor group, are discussed. The various standard of care and experimental techniques as well as protocols for sperm freezing and the advantages and disadvantages of each technique are described in the chapter. The chapter also provides a description of the current challenges and future research directions for sperm cryopreservation.
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The liverwort Marchantia polymorpha has become one of the model organisms, since it has less genetic redundancy, sexual and asexual modes of reproduction and a range of genomic and molecular genetic resources. Cryopreservation of fertile spermatozoa eliminates time, space and labor for growing and maintaining male plants in reproductive phase, and also provides an optional way to backup lines. Here we report a protocol to cryopreserve spermatozoa of M. polymorpha in liquid nitrogen. A cryoprotective solution containing sucrose, glycerol and egg yolk and controlled cooling and warming processes led to successful recovery of motile M. polymorpha spermatozoa after the cryogenic process. The survival rate and average motility of spermatozoa after cryopreservation were maintained at 71 and 54% of those before cryopreservation, respectively. Cryopreserved spermatozoa were capable of fertilization to form normal spores. The technique presented here confers more versatility to experiments using M. polymorpha and could be applied to preservation of plant spermatozoa in general.
Chapter
The basic principles for cryopreservation of sperm are not different from cryopreservation of embryos, eggs, and ovarian tissue. However freezing with sperm is much easier because there is such a minute amount of water in this small, otherwise highly specialized gamete. In fact very little advance has been made in sperm freezing research, because the results seen to be so adequate with the simplest of methods [1–22]. However I will describe, in addition to routine sperm cryopreservation, a simple new method for freezing individual sperm for difficult cases of TESE.
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The objective of this study was to investigate the effect of different levels of L-Carnitine on Ghezel ram semen characteristics after freeze-thawing. Semen samples were collected from 5 healthy and mature rams using an artificial vagina. Different concentrations of 35, 70, 105, mM L-Carnitine were diluted with lecithin-based semen extender and cooled to 5°C and then placed in liquid nitrogen vapor and finally were stored in liquid nitrogen tank. The experiment was carried out on the basis of completely randomized design with 5 replicates. The results showed that the highest total motility and progressive motility were observed in groups containing 70 and 105 mM L-Carnitine that were not significantly different from the control group. The lowest motility recorded in this experiment was the first level of L-Carnitine. In other parameters only VAP, ALH, VCL and LIN were significantly different from the control group. The first level of L-Carnitin was statistically significant in morphology compared to the control group. Addition of 105 mM of L-Carnitine caused higher membrane integrity and lower MDA level compared to 35 mM group (p<0.05). MDA was not significant at any level. Therefore, supplementation of L-Carnitine in lecithin-based semen extender can improve semen quality of Ghezel ram and reduce negative effects of freeze-thawing process.
Article
Conventional sperm freezing procedures need the addition of a relatively large volume of cryoprotectant. The dilution of extremely poor sperm suspensions from ejaculate or testicular tissue may make the recovery of viable spermatozoa difficult at the moment of the intracytoplasmic sperm injection (ICSI) procedure. The cryopreservation of a few spermatozoa in empty zonae pellucidae is an interesting solution for crypto-azoospermic infertile men. We have modified this procedure by filling empty human zonae with TEST Yolk Buffer, an optimal cryoprotective medium, in order to analyse the number of zonae lost after thawing, the number of recovered spermatozoa per zona after thawing, and the sperm motility rate before freezing and after thawing. Fifty empty human zonae pellucidae previously filled with TEST Yolk Buffer were injected with 750 motile spermatozoa from ten infertile men (15 spermatozoa per zona). Sterile straws containing two zonae each were frozen following a two-phase protocol. All of the zonae and 445/750 spermatozoa (59%) were recovered. The mean number (+ SD) of spermatozoa per zona was 8.9 +/- 1.9 (range: 5-12). The recovery rate of motile spermatozoa was 73% (327/445), with a mean number of motile spermatozoa per zona of 6.5 +/- 1.7 (range: 3-10). The cryopreservation of a small number of motile spermatozoa within empty zonae pellucidae using TEST Yolk Buffer as a freezing medium is possible without any major loss of spermatozoa and with an appreciable maintenance of sperm motility. This procedure seems to avoid: i) uncertain sperm retrieval after a laborious and time-consuming search on the day of oocyte aspiration; ii) the need for a repeated testicular biopsy; and iii) the need for heterologous insemination or oocyte cryopreservation (11).
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We examined the damaging effects on spermatozoa of endogenous phospholipid peroxidation brought about by aerobic incubation at 37 degrees C in the presence of 0-5 mM-ascorbic acid and 0-5 mM-FeSO4. As well as becoming immotile, such peroxidized spermatozoa also lost, through leakage, certain intracellular enzymes into the surrounding medium, on a scale resembling that produced by cold shocking non-peroxidized spermatozoa. Morphological observations revealed that peroxidation damaged the plasma membrane, particularly in the region of the acrosome. Further experiments showed that lipid peroxidation irreversibly abolished the fructolytic and respiratory activity of spermatozoa. The susceptibility of spermatozoa to peroxidation was greater when the cells were damaged before incubation with ascorbic acid and FeSO4. To some extent, peroxidation could be prevented, but not reversed, by the addition to sperm suspensions of dialysed egg yolk or dialysed bull seminal plasma. However, dialysed seminal plasma from ram, stallion or man had no protective effect.
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Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.
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To determine whether there is a prognostic value in the percentage normal sperm morphologic features in a human in vitro fertilization (IVF) program, the authors conducted a prospective study in women with bilateral tubal damage. Based on the percentage of morphologically normal spermatozoa, the patients were divided into four groups: group I, normal morphologic features between 0% and 14%; group II, 15% to 30%; group III, 31% to 45%; and group IV, 46% to 60%. One hundred ninety successful laparoscopic cycles were evaluated. In group I, 104 oocytes were obtained, of which 37% fertilized, but no pregnancy resulted; in group II, 81% of 324 oocytes were fertilized, with a pregnancy rate per embryo transfer (ET) of 22%; in group III, 82% of 309 oocytes were fertilized, with a 31% pregnancy rate; and in group IV, 91% of 69 oocytes were fertilized, with a pregnancy rate of 12%. Probability models indicated that there was a clear threshold in normal sperm morphologic features at 14%, with high fertilization and pregnancy rate in the groups with normal sperm morphologic features > 14%.
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Glycerol has commonly been employed as a cryoprotectant in cryopreservation of human spermatozoa. However, the addition of glycerol into the sperm before freezing and the removal of glycerol from the sperm after freezing and thawing result in anisotonic environments to the cells, which can cause cell injury. To define optimal procedures for the addition/removal of glycerol and to minimize the cell injury, one needs to know the kinetics of glycerol permeation across the sperm plasma membrane at different temperatures. For this, one has to determine the permeability coefficient of glycerol (Pg) and its activation energy (Ea). Values of Pg at different temperatures and at different glycerol concentrations were determined by measuring the time required for 50% spermolysis in hyperosmotic glycerol solutions which were hypotonic with respect to electrolytes. Value of the Ea was determined assuming an Arrhenius type temperature dependence of Pg. A dual fluorescent staining technique (propidium iodide and 6-carboxyfluoroscein diacetate) and flow cytometry were used to measure the spermolysis. The values of Pg in 0.5, 1.0, 1.5, and 2.0 M glycerol at 22 degrees C are 1.62, 1.88, 1.68, and 1.54 x 10(-3) cm/min, respectively. The values of Pg in 1 M glycerol at 0, 8, 22, and 30 degrees C are 0.33, 0.54, 1.88, and 2.60 x 10(-3) cm/min, respectively. The value of Ea is 11.76 kcal/mol.
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Human semen was cryopreserved using Human Sperm Preservation Medium, TEST-Yolk buffer, or glycerol alone. Sperm characteristics for each specimen were measured before and after freezing to determine which cryopreservative resulted in better cryosurvival and recovery of motile sperm. Sperm frozen in Human Sperm Preservation Medium had a significantly better recovery of all semen parameters (motility, velocity, and recovery) than either TEST-Yolk or glycerol alone. Statistical analyses also were done to examine the variability between and within donor semen specimens. Differences between donors, between specimens, and measurements within donors all contributed to variability of sperm characteristics. Specimen-to-specimen variability for a given donor represented 12% to 47% of the total variability, whereas processing and measurement variability represented 12% to 41%. Donors also varied in the ability of their sperm to tolerate freezing. There was a relationship between motile count after dilution with cryopreservative and post-thaw motile count. This relationship allows the prediction of poor-thaw survival before freezing a specimen.
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Cells subjected to the events occurring before, during, and after freezing and thawing are exposed to major changes in the osmotic pressure of the surrounding medium; i.e., the osmolalities can exceed 30. An important question in understanding the mechanisms of injury is whether cells respond as ideal osmometers to these strongly anisotonic solutions. Mouse and bovine embryos from eight-cell to blastocyst stage were used to investigate the question. They were found to behave as ideal osmometers at room temperature over a wide range of tonicities; i.e., from four times isotonic to almost 1/3 times isotonic, ideality being defined by a Boyle-van't Hoff equation. Embryo volumes increased from 40 to 200% of isotonic over this range and survivals of mouse embryos were unaffected. However, outside this range the membrane apparently becomes leaky and the survival of mouse embryos drops sharply. Osmolalities rise to high values during freezing and the paper develops the thermodynamic equations to show how computed cell volumes as a function of subzero temperature can be translated into the Boyle-van't Hoff format of cell volume as a function of the reciprocal of osmolality.
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The records of 227 couples who received artificial insemination by frozen donor semen in 984 treatment cycles were analysed to find out the factors affecting the pregnancy rates. The cumulative pregnancy rate at 6 months was 46.8% and the pregnancy rate per cycle was 10%. The pregnancy rates were adversely affected by (1) increase in the recipient age above 30 years, (2) irregularity of menstrual cycles, (3) the use of clomiphene citrate for induction of ovulation, and (4) low cervical mucus scores. Among our donor semen samples selected with a set of criteria, the sperm counts and initial and post-thaw motility did not affect the pregnancy rates.
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In at least 4 of 7 cases, fertilization of intact human oocytes was more successful when spermatozoa were pretreated with TEST yolk medium at 5 degrees C for 2 hours as compared with the standard treatment with Ham's F-10 only. Both pregnancies that were obtained after the transfer of the fertilized oocytes resulted from oocytes fertilized by TEST yolk-treated spermatozoa. No decrease in fertilization occurred in any of the cases after TEST yolk treatment. If these results hold true for a larger series of patients, it may be worthwhile for the standard IVF incubation system of spermatozoa to include TEST yolk.
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Penetration of zona-free hamster oocytes by human spermatozoa after preincubation with BWW medium (standard technique) or a buffer system containing egg yolk (TESTY) was compared by applying both treatments to aliquots of 60 ejaculates from 34 patients. The TESTY-treated spermatozoa penetrated the oocytes much more successfully than the spermatozoa treated by the standard technique. Although the penetration levels achieved by the two methods were significantly correlated, the correlation coefficient was too low to predict the penetration outcome from one technique to the other. More consistent results were obtained when different ejaculates from the same donor were treated by TESTY than by the standard technique. Spermatozoan penetrating ability was more strongly correlated with sperm concentration, motility, and morphology after treatment by the standard technique than after TESTY treatment. Much larger differences in penetration were present between normal and abnormal ejaculates when the standard technique was used than TESTY.
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Sperm survival and in vitro fertilizing capacity were examined following cryopreservation with three concentrations of glycerol (0, 2, and 7.5%) and four cryopreservation media. Post-thaw motility and motility index increased with increasing concentrations of glycerol. Post-thaw velocity, linearity, percentage of zona-free hamster oocytes penetrated, and penetrations per oocyte were greater following freezing with 2 or 7.5% glycerol than with 0% glycerol. A significant interaction between media and glycerol was observed for motility, velocity, linearity, motility index, and percentage of oocytes penetrated. These results suggest that the concentration of glycerol required for optimal survival and in vitro fertilizing capacity of human sperm following cryopreservation is dependent on the type of medium used for freezing.
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A series of experiments was conducted to examine potential toxic effects of cryoprotectants on motility of human spermatozoa. The data indicated that exposure of spermatozoa to cryoprotectant medium for as little as 15 minutes at room temperature caused a reduction in motility. This reduction in motility was caused by glycerol. Lowering glycerol concentrations from 7.5% to 5.0% improved sperm motility at 24 hours post-thaw. Sperm motility was not affected by either slow or abrupt cooling rates above -5 degrees C. Motility was greater in cryopreserved sperm at 24 hours post-thaw when glycerol was added at -5 degrees C rather than at room temperature. These data suggest that avoiding glycerol toxicity either by reducing the concentration used or by adding glycerol at a lower temperature, or both, may improve human sperm cryosurvival rates.
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Optimization of each step in the standard protocol of the sperm penetration assay has increased the sensitivity of the traditional assay, and this increased sensitivity has allowed us to avoid the production of false-positive results. Development and use of routine quality control methods now enable us to compare different samples from a given individual and monitor with confidence biological variation and response to medical or surgical treatment. Although our test is extremely predictive of how well a patient's specimen may do as a result of normal intercourse, artificial insemination, and human in-vitro fertilization, it must be stressed that the sperm penetration assay and the sperm capacitation index reported by our laboratory only measure the ability of a sperm to capacitate and fuse with zona-free hamster ovum. How well spermatozoa disperse the cumulus and bind to and penetrate the human zona pellucida is not measured by this test. This would be reflected by an apparent "false-positive" outcome in the sperm penetration assay. However, our data from 138 patients suggest that this occurs only on rare occasions (in 2 of the 138). The test can be used only as a supplement to a conventional semen analysis because other factors, such as motile concentration and motility characteristics, clearly are also of importance particularly in vivo. The results obtained by our laboratory using the optimized assay described herein demonstrate that this test can play an important role in the evaluation of an infertile man.
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Several cryoprotective media currently in use for human sperm cryopreservation have been directly compared with each other by use of a static vapor freezing technique. The TEST-citrate-yolk buffers with glycerol resulted in the highest motility and longevity after thawing; thus, their use is recommended.
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PIP The history of frozen human semen is reviewed and the clinical devel opments which began in 1964 are given in greater detail. Artificial insemination has become widely accepted and thousands have been performed successfully. 40-45% survival of spermatozoa have been recorded after 10 years of storage. No evidence suggests that storage produces genetic changes. Conservative data show 564 children have been born from frozen semen using approved insemination procedures, 7 abnormal children were born, 50 spontaneous abortions were recorded, and 65 pregnancies had not yet resolved themselves. Periods of preservation ranged from 13 months (Japanese series) to 10 1/4 years (U.S.). Names and addresses of researchers maintaining semen banks are given. Application of the technology to infertility, as "insurance" for men undergoing vasectomy, and as a method for improving genetic characteristics are discussed. Commercial banks with regulated standards and a clarified policy of no guarantee of fertility were endorsed as essential to large-scale application of this technology.
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Der Einfluß von kryprotektiven Medien und Verdünnungsmedien auf die Erhaltung menschlicher Spermatozoen Menschliche Spermatozoen-Erhaltungs-Medium (HSPM), das aus 15% Glyzerin, 0,05 M Sucrose und 1% Glycin besteht, hat eine gleichwertige oder sogar bessere Kapazität die Spermatozoen-Motilität und - Vitalität zu erhalten als Eigelbzitratmedium (ECM), welches 15% Glyzerin enthält. Die Inseminationen mit dem Sperma, das in HSPM eingefroren wurde, hatten eine höhere Schwangerschaftsrate als wenn das Sperma in ECM eingefroren war. Glyzerin war DMSO oder Aethylenglykol als Kryoprotektants überlegen, obwohl die kryoprotektive Fähigkeit von 7,5% Glyzerin und 7,5% Aethylenglykol nicht signifikant differierten. Weder die Zusatz-Temperatur noch die Methode des Zusatzes der Medien, die Glyzerin, DMSO oder Aethylenglykol enthielten, hatte irgendeinen Einfluß auf die Sper-matozoen-Überlebensart bei Tiefgefrierung. Raffinose (0,025 bis 0,05 M) oder Sucrose (0,05 bis 0,1 M) besserten die Überlebensrate der eingefrorenen Spermatozoen. Der pH von HSPM hatte keinen signifikanten Einfluß auf die Überlebensrate der tiefgefrorenen Spermatozoen; der ideale pH lag zwischen 6,5 und 7,0. Das Verhältnis von 1:1, 1:2, 1:3 und 1:4 des Verdünnungsmediums zum Sperma hatte keinen Einfluß auf die Überlebensrate der tiefgefrorenen Spermatozoen.
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This study compared the cryoprotective effect of glycerol with that of a zwitter ion buffer system (TESTCY). Spermatozoa that are cryopreserved in the presence of glycerol possess a somewhat higher progressive motility immediately after thawing than those preserved in the presence of TESTCY. However, after a 1-hour incubation in glycerol-free medium, the progressive motilities of the glycerol- and TESTCY-treated spermatozoa become essentially identical. After 2 hours in culture medium, TESTCY-treated spermatozoa possess a higher motility than glycerol-treated spermatozoa, indicating that TESTCY is a better preservative than glycerol for the long-term motility of human spermatozoa. The fertilizing potential of the cryopreserved spermatozoa was assessed by their ability to penetrate zona-free hamster oocytes in vitro. Spermatozoa that are cryopreserved in the presence of TESTCY produce three- to four-fold higher penetration rates than glycerol-treated, cryopreserved spermatozoa. Cryopreservation in the presence of TESTCY also results in a higher stability of the acrosin/proacrosin system than when the spermatozoa are preserved in glycerol, since about two- to three-fold higher levels of proacrosin are retained. These results indicate that TESTCY is a better cryopreservative for human spermatozoa than glycerol.
Article
The efficacies of fresh versus cryopreserved semen in the treatment of male factor infertility by artificial insemination by donor (AID) semen were directly compared by using the patient as her own control. In any one cycle, either fresh or frozen semen was used. The type of semen preparation was randomly assigned for the first cycle and varied thereafter according to donor availability. The same donor was used for a given patient in six consecutive cycles. We treated 381 patients in this way. In 676 cycles fresh semen was used and 128 pregnancies were achieved. Fecundability, the chance of getting pregnant per cycle of exposure, was 18.9% with fresh semen. In 1200 cycles cryopreserved semen was used and 60 pregnancies occurred, for a fecundability of 5.0%. Therefore, in our clinic, fresh semen is more than three times as likely to induce pregnancy as frozen semen. The design that has been used in this therapeutic protocol provides a technique for internal quality control of the cryopreservation process and for the investigation of other variables potentially affecting the success rates with AID.
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The ability of human spermatozoa to penetrate zona-free hamster ova was examined following low-temperature capacitation (4 degrees C) in TES-Tris (TEST)-yolk buffer for periods of up to 66 hours. Results obtained from 66 individuals demonstrated that the number of penetrations per ova were increased by an average of 2.5-fold when spermatozoa were capacitated for 42 hours, as compared with 18 hours. Furthermore sperm with extremely poor penetration rates after 18-hour capacitation was often improved by longer capacitation periods. Patients from our infertility clinic with less than 20 X 10(6) spermatozoa/ml of ejaculate had significantly lower penetration rates when compared with patients and donors with greater than 20 X 10(6) spermatozoa/ml (P less than or equal to 0.001). The observed effects of TEST-yolk buffer appear to be related to its ability to preserve sperm motility over prolonged periods, during which increased capacitation of the total sperm population is achieved.
Article
Cryopreservation of semen is associated with reduced motility after thawing, resulting in a decreased pregnancy rate. Artificial stimulation of motility has been used in fresh semen samples. This study measured the effect of motility stimulants on various motion characteristics and other sperm functions using cryopreserved semen. Frozen semen samples from healthy donors were thawed, and motility stimulants were added in vitro and incubated for 60 minutes. The percentages of motile spermatozoa in each specimen and other motion characteristics were measured. In addition, spermatozoon's viability, membrane integrity, and ability to penetrate bovine cervical mucus were studied after addition of stimulants. Percentage motility and all other motion characteristics improved after stimulation with pentoxifylline, caffeine, or 2-deoxyadenosine. Linearity did not significantly differ in the control samples after adding any of the stimulants. Viability, membrane integrity, and penetration ability did not improve significantly and were comparable with control values. Pentoxifylline, caffeine, and 2-deoxyadenosine can stimulate sperm motility and other motion characteristics. This may be beneficial in the cryopreservation of sperm from normal donors and oligozoospermic patients for use in assisted reproduction.
Article
The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.
Article
Media prepared with egg yolk and two buffers, TES and Tris, called TEST-yolk have been shown to have beneficial effects on the survivability, fertilizing capacity and storage potential of human spermatozoa. Egg yolk lipoproteins are the critical compounds for the beneficial effects, with a synergistic effect due to the TES-Tris buffer component. TEST-yolk media have been used in the sperm penetration assay, the hemizona assay, sperm preparation for clinical in-vitro fertilization, artificial insemination with homologous spermatozoa, cryopreservation, sperm samples with a positive antisperm antibody test, and preparation for techniques requiring capacitated spermatozoa. Few harmful consequences of TEST-yolk have been reported, although controlled trials are required to evaluate therapeutic effects in the treatment of male infertility.