Lipid dynamics in the plasma membrane of fresh and cryopreserved human spermatozoa.

Preserving the integrity of the plasma membrane of spermatozoa is crucial for retention of their fertilizing capacity, especially after stressful procedures such as freezing and storage. In this investigation we have measured lipid diffusion in different regions of the plasma membrane of fresh and cryopreserved human spermatozoa using a sensitive, high resolution fluorescence photobleaching technique (FRAP) with 5-(N-octadecanyl)aminofluorescein as reporter probe. Results show that diffusion was significantly faster on the plasma membrane overlying the acrosome and decreased progressively in the postacrosome, midpiece and principal piece. The midpiece plasma contains a higher proportion of immobile lipids than other regions. In cryopreserved spermatozoa, lipid diffusion in the plasma membrane was significantly reduced on the acrosome, postacrosome and midpiece relative to fresh spermatozoa. Diffusion, however, could be restored to normal levels by washing spermatozoa in a medium containing 0.4% polyvinylpyrrolidine but not in medium alone or in medium containing 0.4% albumin. These results suggest that (i) lipid dynamics in the plasma membrane of human spermatozoa varies significantly between surface regions; (ii) in-plane diffusion is adversely affected by cryopreservation; and (iii) washing frozen spermatozoa in 0.4% polyvinylpyrrolidine restores membrane lipid fluidity to normal levels. The latter finding has important implications for improving the fertility of human spermatozoa following cryopreservation.     

Case report: Changes in motility patterns during in-vitro culture of fresh and frozen/thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection

The present report describes the motility changes in vitro (percentagemotile and progressively motile) of freshly collected testicularand epididymal spermatozoa and following freeze/thaw of thesame spermatozoa from a man with obstructive azoospermia. Washedspermatozoa were cultured in micro droplets under paraffin oilor in test tubes using HEPES-buffered or bicarbonate-bufferedmedium containing 10% human serum. In fresh testicular spermcultures 60–65% of the sperm cells became motile within2 days of culture; the motility was maintained for a further4–5 days before a decline was observed. The progressivemotility unproved markedly on the third day of culture and itpeaked around day 5. Only a small number of frozen/ thawed testicularspermatozoa became motile during in-vitro culture (15–20%)and the motility was maintained for only 2–3 days beforeit declined. Furthermore, only 10–12% of the spermatozoashowed progressive motility. Spermatozoa recovered from micro-epididymalsperm aspiration (MESA) showed a gradual decrease in progressivemotility and in 5 days all sperm cells were found to be immotilein both freshly collected and frozen/thawed spermatozoa. Allculture systems supported sperm motility. It is clear that testicularspermatozoa, particularly from men with obstructive azoospermia,can be collected and maintained in vitro for up to 1 week beforethe oocyte retrieval but when frozen testicular or epididymalspermatozoa are used it is more reliable to thaw these spermatozoaon the day of intracytoplasmic sperm injection.     

A novel approach to sperm cryopreservation

Human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during cryopreservation are all non-linear with time. In this paper it is shown that improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes instead of imposing a linear temperature reduction. Several treatments were compared: control of solidification to achieve constant ice formation with time was more damaging than the standard linear reduction in temperature. However, treatments which followed a chosen non-linear concentration profile, referred to as 'controlled concentration' allowed recovery of almost all the cells which were motile before freezing. The biophysical basis of these different responses was examined using the cryostage of a scanning electron microscope and freeze substitution and it was found that, surprisingly, all samples of spermatozoa in the frozen state were neither osmotically dehydrated nor had any visible intracellular ice. Viability on thawing did not appear to correlate with conventional theories of cellular freezing injury, which suggests that for human spermatozoa other factors determine viability following freezing and thawing.     

The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze-thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen-thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen-thawed populations. It appears feasible to use frozen-thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.     

Cryopreservation of spermatozoa: a 1996 review

Both the ability to freeze human spermatozoa and the possibilityof pregnancy following intrauterine insemination have existedfor >40 years. There have been a number of improvements duringthat time concerning the methods of freezing and thawing humanspermatozoa. Initially, the use of the cryoprotective propertiesof glycerol allowed a major improvement; subsequently, changeswere mainly empirical. It was a long time before specific cryobiologicalstudies were undertaken. However, the necessity for these becameapparent with the partial recovery or sometimes loss of motilityafter freezing either subfertile semen before chemotherapy orradiotherapy, or spermatozoa collected from non-physiologicalsituations (epididymal or testicular spermatozoa). The maintrends in improvement have defined end-points other than thepercentage of motility recovery or the assessment of ultrastructuraldamage. More sensitive criteria of the objective assessmentof motility, energy status, damage to the plasma membrane orto subcellular elements, chromatin stability and chromosomaldamage have been proposed as complementary end-points to betterassess sperm cryopreservation. A different approach was relatedto the biochemical environment and physical conditions imposedon spermatozoa during the freezing and thawing process. Biochemicalchanges were assessed following different combinations of variousextenders which attempted either to better preserve some parameteror to avoid the tendency towards drastic increase in osmoticpressure. Analysis of physical conditions was linked to therate of cooling, freezing ad warming, and was based on cryobiologicalstudies. Finally, even though such improvements are not negligible,many questions remained unanswered. The extensive use of frozenspermatozoa during assisted reproductive techniques, togetherwith the development of assisted fertilization using surgicallycollected spermatozoa, creates the need for additional studiesto improve the cryopreservation of human spermatozoa. Keywords: cryopreservation/review/spermatozoa     

Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation (95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men.     

Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate

Cryopreservation causes extensive damage to spermatozoa, thereby impairing their fertilizing ability. The purpose of this study was to determine if the direct addition of pentoxifylline to the seminal plasma before cryopreservation improved sperm motility and acrosome reaction. Semen specimens from 15 healthy volunteers were divided into two aliquots. One aliquot was treated by adding 5 mM pentoxifylline directly to the seminal plasma (treatment group) and the other aliquot received no treatment (control group). Both aliquots were then cryopreserved by using the liquid nitrogen freezing method. The percentage of motile spermatozoa and various motion characteristics were then evaluated by performing computer-assisted semen analysis. The sperm viability was determined with a supra-vital dye, Hoechst-33258, and the acrosome reaction (spontaneous and calcium ionophore-induced) was monitored using fluorescein isothiocyanate-conjugated peanut lectin (FITC-PNA) binding assays. Pentoxifylline treatment significantly increased the sperm motility, the amplitude of lateral head displacement, the hyperactivation status, and the frequency of spontaneous acrosome reactions before freezing (P < 0.05). After post- thaw, no difference in motion characteristics (except percentage motility) between treated and control groups were observed. Acrosome loss due to the freeze-thaw process was less in the pentoxifylline- treated group (P = 0.0003). In addition, the percentage of cryopreserved acrosome-intact spermatozoa that underwent further acrosome reactions in response to calcium-ionophore challenge was significantly higher in the treated group (P = 0.03). Pentoxifylline treatment before freezing improved the acrosome reaction to ionophore challenge in cryopreserved spermatozoa. Treatment with pentoxifylline appears to minimize sperm damage during the freeze-thaw process and may improve fertilization rates with assisted reproductive procedures such as intrauterine insemination or in-vitro fertilization.      

Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage

When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Therefore, annexin V-binding, in conjunction with flow cytometry, was used to evaluate the integrity of the sperm plasma membrane after different cryostorage protocols: i.e. 10% (v/v) glycerol; sperm maintenance medium (MM); freezing medium TEST yolk buffer (TYB); or cryostorage without protection (cryoshock). Using a combination of two fluorescent dyes, annexin V and propidium iodide (PI), led to three groups of spermatozoa being identified: (i) viable spermatozoa (annexin V-negative and PI-negative); (ii) dead spermatozoa (annexin V-positive and PI-positive); and (iii) cells with impaired but integer plasma membrane (annexin V-positive and PI-negative). The percentage of vital annexin V-negative spermatozoa increased significantly (P < 0.05) from spermatozoa treated by cryoshock (15.0+/-1.2%) to spermatozoa cryopreserved by TYB (26.6+/-2.2%) via cryopreservation by 10% (v/v) glycerol (19.9+/-1.6%) and by MM (22.2 1.8%) and was associated with the percentage of motile spermatozoa (17.6+/-3.4% by glycerol; 19.6+/-3.7% by MM and 22.6+/-3.9% by TYB; P = 0.0001). Of the spermatozoa, 12-22% were annexin V-positive even though they did not bind to PI, indicating viability before as well as after cryostorage. The percentage of vital annexin V-positive spermatozoa was significantly correlated with different sperm motility parameters (velocity straight linear, r = 0.601, P = 0.018; percentage of linearly motile spermatozoa: r = 0.549, P = 0.034). We, therefore, concluded that annexin V-binding is more sensitive in detecting a deterioration of membrane functions than PI staining, and that a considerable percentage of spermatozoa might have dysfunctional plasma membranes besides dead or moribund cells. Of the cryopreservation protocols tested, TYB yielded the most viable spermatozoa. Therefore, we advocate the use of the annexin V-binding assay for the evaluation of the quality and integrity of spermatozoa.     

Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing

The purpose of this study was to examine if selecting a sperm population with improved motion characteristics before freezing reduces the deleterious effects of cryopreservation. Semen specimens from 15 normal donors were divided into two equal aliquots. The first aliquot received no treatment (control), and the second was processed by swim-up from a washed sperm preparation to select a sperm population with better motility and motion characteristics (swim-up). Both aliquots were cryopreserved by the liquid nitrogen vapour method. Percentage motility and motion characteristics were evaluated by computer-assisted semen analysis. Acrosome integrity as well as spontaneous and calcium ionophore-induced acrosome reactions before freezing and after thawing were assessed by fluorescein isothiocyanate conjugated peanut agglutinin combined with a supra vital dye (Hoechst-33258). Swim-up processing enabled selection of a sperm population with better motion characteristics, percentage motility and viability before freezing (P < 0.001), but with no difference in percentage of acrosome-intact spermatozoa (P = 0.63). After thawing, the swim-up specimens exhibited faster velocity and progression than untreated specimens (P < 0.001). They also had higher percentages of spermatozoa with intact acrosomes and spermatozoa able to undergo acrosome reaction in response to calcium ionophore (P < 0.05). Selecting a highly motile sperm population before freezing enhances overall post-thaw spermatozoa quality.     

Fertilization and early embryology: Direct assessment of cryopreservation of human spermatozoa using a cryomicroscope and computer-aided sperm analysis

Use of a cryostage has enabled direct observation of human spermatozoaas they are cryopreserved and thawed. Crystallization and recrystallizationevents are readily observed. In combination with computer-aidedsemen analysis (CASA) equipment it was possible to determinethe consequence of altering the cooling, freezing and thawingrates of a temperature-rate profile on sperm motility. Increasingthe cooling rate to 50°C/min resulted in significantly lowerpre-freeze to post-thaw ratios for average path velocity (VAP,13%), mean straight line velocity (VSL, 35%), mean linearity(LIN, 28%) and straightness (STR, 24%), while the ratio of thenumber of cells crossing the field of view (NCF) significantlyincreased (30%) compared to a standard freeze-thaw temperaturerate profile. The NCF pre-freeze to post-thaw ratio was associatedwith the percentage of cell recovery after cryopreservation.Faster thaw rates resulted in better survival of the cells,perhaps due to the shorter time during which recrystallizationoccurred. The NCF ratios were significantly higher (33 and 30%for thaw rates of 50 and 100°C/min respectively) than forthe standard profile samples. Previous studies on cell survivalhave shown a link between the cooling and thaw rates. The cryostageshould prove invaluable in future studies to identify the causesof cryodamage to spermatozoa. When used in combination withCASA, changes to sperm function during cryopreservation canbe accurately measured.     

Cryopreservation of single human spermatozoa

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.      

Cryopreservation of fractionated, highly motile human spermatozoa: effect on membrane phosphatidylserine externalization and lipid peroxidation

INTRODUCTION: This study investigated lipid peroxidation (LPO) and membrane integrity following cryopreservation-thawing. METHODS: Infertile men (study group) and donors (control group) were examined. Purified populations of highly motile spermatozoa were cryopreserved using TEST-yolk buffer and glycerol (TYB-G) followed by quick thaw. LPO was measured by a spectrophotometric assay, with and without a ferrous ion promoter. Annexin V binding was used to assess membrane translocation of phosphatidylserine (PS). RESULTS: Pre-freeze LPO was significantly higher in the study than in the control group (P = 0.03). In both groups, LPO measurements after thawing were significantly higher than the pre-freeze samples not exposed to TYB-G (P = 0.002 and P = 0.001 respectively). However, when the pre-freeze samples with TYB-G were compared with the post-thaw samples (all exposed to TYB-G), these differences were not significant. There was a significant increase in PS externalization following cryopreservation in both groups (P = 0.02 and P = 0.003 respectively). In donors, pre-freeze LPO concentrations had a significant positive correlation with thawed spermatozoa depicting PS externalization (r = 0.77, P = 0.04). CONCLUSION: Although patients had higher basal LPO than donors, LPO did not differ between fresh and cryopreserved-thawed fractionated motile spermatozoa. Freezing-thawing was associated with translocation of PS to the external membrane leaflet.     

Effects of cryopreservation on progesterone-induced ion fluxes and acrosome reaction in human spermatozoa

The present study evaluated the effects of cryopreservation on progesterone-induced variations of calcium ion concentration [Ca(2+)](i), plasma membrane potential and acrosome reaction in human spermatozoa. Spermatozoa from 10 fertile donors were divided in two equivalent aliquots, one used as control (fresh spermatozoa) and the other used after freezing-thawing. Measurement of spermatozoa [Ca(2+)](i) before and after freezing-thawing showed a significant reduction of basal [Ca(2+)](i) in thawed spermatozoa (P < 0.01). Progesterone induced a rise of [Ca(2+)](i) both in fresh and thawed spermatozoa with a significant reduction after freezing-thawing (P < 0.01). The monitoring of sperm plasma membrane potential demonstrated that progesterone induced plasma membrane depolarization in fresh spermatozoa that was absent in thawed spermatozoa. The inhibitory effects of freezing-thawing on progesterone induced [Ca(2+)](i) and plasma membrane potential variations in human spermatozoa were closely related to the inhibition of the acrosome reaction. In conclusion the present study demonstrates that freezing-thawing procedures reduce the responsiveness of human spermatozoa to progesterone in terms of [Ca(2+)](i) rise and completely inhibit its effects on plasma membrane potential variations, thus supporting the hypothesis that freezing-thawing procedures may differently modify the plasma membrane receptors for progesterone in human spermatozoa which are known to express at least two receptors for this steroid in their plasma membrane.     

Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime

While washing of human sperm cells by centrifugation and resuspensionis a procedure in widespread use, there have been indicationsthat this procedure per se may be harmful to the cells. Theobjective of this study was to investigate this question. Tothis end, a method for the clean separation of motile humanspermatozoa from seminal plasma in the absence of centrifugationwas developed, using a modified swim-up procedure, in whichliquefied semen was mixed with an equal volume of 30 mg/ml dextranin medium, and the mixture overlaid with medium containing 5mg/ml bovine serum albumin, forming two discreet layers withstable interface. The percentage of motile cells in a givensample was consistently >80% immediately after recovery.Damage to the cells was assessed by loss of motile cells duringincubation up to 96 h post-recovery. Comparison of aliquotsof spermatozoa obtained by the dextran swim-up procedure showedthat the aliquot subjected to centrifugation had 4 ±3% motile cells after 48 h, while the untreated aliquot had52 ± 12%. The aliquots showed no difference 1 h postrecovery.Similar results were obtained with spermatozoa that had beencentrifuged in seminal plasma and resuspended in fresh plasma,then recovered by dextran swim-up. The delayed onset of motilityloss in the centrifuged samples implies that this treatmentinduces sublethal damage in the cells. Comparison of the standardswim-up and Percoll gradient methods for sperm recovery, bothof which involve centrifugation steps, showed decline in motilityof the samples similar to that seen with dextran swim-up ofcentrifuged cells. We conclude that centrifugation per se inducessublethal damage in human spermatozoa, independently of treatmentmethod, and suggest that recovery methods for human spermatozoawhich avoid centrifugation might partially alleviate the damageincurred by these cells during cryopreservation.     

Testicular sperm extraction (TESE) and ICSI in patients with permanent azoospermia after chemotherapy

BACKGROUND: Patients persistently azoospermic after chemotherapy have been considered traditionally as sterile unless sperm was frozen before therapy. Recent advances during the last decade combining testicular sperm extraction (TESE) and ICSI in patients with non-obstructive azoospermia allow these males to father their own genetic offspring. METHODS: A retrospective study was conducted of 12 patients with non-obstructive azoospermia after chemotherapy undergoing TESE between 1995 and 2002. Cancer type and anti-neoplastic treatments were recorded, together with maximum testicular volume, serum FSH levels and testicular histopathology. When TESE was successful, spermatozoa were cryopreserved for performing ICSI later. RESULTS: In five patients (41.6%) motile spermatozoa for cryopreservation and ICSI were retrieved. Four of them had received chemotherapy for testicular cancer, and one had been treated by chemotherapy/radiotherapy for Hodgkin's disease. Clinical and histological parameters were unable to predict with certainty TESE outcome in an individual patient. Eight ICSI cycles were performed on five couples and one pregnancy was obtained which resulted in the delivery of a healthy girl. CONCLUSION: Some patients with permanent azoospermia after chemotherapy can be successfully treated by TESE-ICSI. This procedure, however, may have potential genetic risks. Therefore, freezing semen before starting gonadotoxic therapy is the strategy of choice, and patients should be counselled accordingly.     

Changes in carnitine and acetylcarnitine in human semen during cryopreservation.

L-Carnitine and acetylcarnitine concentrations were determined in spermatozoa and seminal plasma from 15 men, in both fresh ejaculate and frozen-thawed semen with cryoprotective medium. Sperm motility was also evaluated. In fresh samples, the levels of carnitine and acetylcarnitine in seminal plasma were comparable whereas in spermatozoa, acetylcarnitine predominated. Cryopreservation did not change the carnitine and acetylcarnitine levels in seminal plasma nor the carnitine concentration in spermatozoa; by contrast, the acetylcarnitine level in spermatozoa was decreased in 14 cases (110 +/- 8 versus 210 +/- 20 nmol/10(8) cells). This decrease in acetylcarnitine content was greater during semen dilution in cryoprotectant than after the freezing/thawing process. Motility was also decreased in all cases after the freezing/thawing process. These results suggest that acetylcarnitine recovery in spermatozoa is further evidence of the deleterious effect of the cryoprotective medium in the cryopreservation of semen.     

The first births and ongoing pregnancies associated with sperm cryopreservation within evacuated egg zonae

This new procedure principally aims to avoid a second or possibly multiple surgical procedures for sperm extraction from the male partner in cases of limited amounts of sperm cells, where normal freeze-thaw protocols would fail. Patients (n = 34) diagnosed as azoospermic, extreme oligozoospermic, or oligoasthenozoospermic underwent the process of sperm cryopreservation within evacuated egg zonae. Other samples were allocated to conventional sperm freezing. Sperm samples were acquired using testicular sperm extraction (TESE), microepididymal sperm aspiration (MESA), or fresh ejaculate. Subsequently, five of these 34 couples have undergone in-vitro fertilization (IVF) and achieved normal fertilization using post-thawed spermatozoa frozen under zonae pellucidae in conjunction with intracytoplasmic sperm injection (ICSI). The average fertilization rate for the post-thaw injected spermatozoa was 65%. This is comparable with the regular fertilization rate of 65% for combined MESA and TESE using fresh spermatozoa. All patients underwent embryo transfer. The average implantation rate per embryo was 31%; nearly the same for regular MESA/TESE ICSI cycles (32%). The first pregnancy associated with this procedure concluded with the full term delivery of healthy twin girls on July 18, 1997. The remaining four thaw procedures resulted in another twin delivery, an ongoing singleton gestation, a negative pregnancy test and a biochemical pregnancy respectively.     

Comparative analysis of motility characteristics of Percoll-selected spermatozoa populations from fresh and cryopreserved semen

The proportion and quality of motility of spermatozoa in normozoospermicejaculates were assessed using computerassisted semen analysis.The ejaculate was split and the motility re-assessed followingseparation on a Percoll gradient with or without cryomediumand cryopreservation. Cryopreservation caused a significantdecrease in the proportion of motile spermatozoa and in theirvelocity and amplitude of lateral head displacement. The initialdecrease in the proportion of motile spermatozoa was found tobe in part an effect of the cryomedium. The use of Percoll gradientseparation did not initially change these effects but after4 h incubation differences in velocity and amplitude of lateralhead displacement between samples were no longer evident. Percoll-selected,cryopreserved spermatozoa had both a stable proportion of motilespermatozoa and a stable velocity for at least 48 h, whereasin fresh spermatozoa populations, similarly separated usingPercoll, the proportion of motile spermatozoa had decreasedby 24 h and the velocity was lower at 48 h. Percoll preparationis an effective method for the selection of motile spermatozoafrom cryopreserved semen which, after a short incubation, havesimilar motility characteristics to fresh spermatozoa.     

Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction.

Several techniques have been used for selecting motile spermatozoa including Percoll and albumin gradients, swim-up, and glass wool filtration. A high yield of motile spermatozoa as well as an enhancement of motility are the most desirable features of a practical method. An equally important consideration is whether or not these techniques select functionally normal spermatozoa. In this study we have compared two methods for separation of motile cells, swim-up and Percoll gradient. Normal semen samples from 12 different men were used in this study. Each sample was simultaneously processed by swim-up and Percoll gradient using modified Tyrode's medium. After the sperm concentration was adjusted to 1 x 10(7) spermatozoa/ml, the suspensions were incubated at 37 degrees C, 5% CO2 in air. In each suspension the percentage of sperm recovery, percentage of motile spermatozoa, percentage of acrosome reacted spermatozoa (either spontaneously or stimulated with human follicular fluid), percentage of zona-free hamster oocytes penetrated, and number of spermatozoa bound to the human zona pellucida were determined. The results obtained indicated that the percentage of sperm recovery was higher with the Percoll gradient than with the swim-up procedure (P less than 0.001). However, no significant differences were found between these two sperm populations in the percentage of motile cells, in the percentage of acrosome reacted spermatozoa, and in the percentage of zona-free hamster oocytes penetrated. In addition, the number of spermatozoa bound per zona pellucida was similar for spermatozoa selected by Percoll or swim-up. We conclude that there were no functional differences between the spermatozoa selected by either method.     

Effects of cryopreservation on human sperm acrosomes.

Total acrosin activity and acrosomal status were determined before and after cryopreserving human spermatozoa. Three different cryopreservation protocols were used. Both acrosin activity and the incidence of intact acrosomes decreased during cryopreservation. The magnitudes of the decreases were weakly but significantly correlated (r = 0.29, P less than 0.05), suggesting that acrosomal loss contributed to the decrease in acrosin activity. The effects of the three cryopreservation protocols were not significantly different. Motility decreased more (average 43%) than did the percentage of spermatozoa with intact acrosomes (27%) and the total acrosin activity (24%). These measurements suggested that acrosomal damage may have been secondary to cell death. This hypothesis was tested by determining the acrosomal status of spermatozoa that survived cryopreservation. Spermatozoa that were motile after thawing averaged 96% acrosome-intact; their acrosin activity, however, was significantly less than that of motile, unfrozen spermatozoa. These observations support the idea that the acrosomal loss due to cryopreservation is associated with cell death but also demonstrate decreased total acrosin activity of the acrosome-intact spermatozoa that survive cryopreservation.