Evaluation of human sperm function after repeated freezing and thawing.
TL;DR: The numbers of normal vital motile sperm after 3 serial freeze-thaw cycles are adequate for bringing about fertilization via intracytoplasmic sperm injection in ART programs.
Abstract: Sperm storage via freezing has been useful for men who have difficulty masturbating during assisted reproductive technology (ART) programs and before impotency caused by chemotherapy, vasectomy, and other procedures. Studies were undertaken to evaluate the extent of cryoinjury to sperm after repeated freezing and thawing. The results showed that normozoospermic and oligozoospermic sperm survived after 3 repeated freeze-thaw cycles. The inclusion of seminal plasma did not seem to protect human sperm during freezing and thawing. There were no significant differences in recovery percentages for motile, vital, and morphologically normal sperm between slow and rapid freezing methods in thaws 1, 2, and 3 of normozoospermic and oligozoospermic unwashed (u), washed (w), and washed + seminal plasma (ws) samples. However, there were significant percentage drops in the recovery of motile and vital sperm between each thaw (ie, first to second thaw, and second to third thaw) using both slow and rapid freezing for u, w, and ws samples (P < .01). There were also no significant differences in percentage recovery of motile, vital, and morphologically normal sperm between u, w, and ws samples during thaws 1 to 3 in the normozoospermic and oligozoospermic groups. Sperm were capable of fertilizing hamster oocytes microinjected with single sperms after 3 freeze-thaw cycles as evidenced by the formation of 2 distinct pronuclei and 2 polar bodies in 22.2% and 17.2% of normozoospermic and oligozoospermic samples, respectively. The numbers of normal vital motile sperm after 3 serial freeze-thaw cycles are adequate for bringing about fertilization via intracytoplasmic sperm injection in ART programs. Thus, leftover washed sperm in laboratories that perform in vitro fertilization can be frozen, thawed, and refrozen several times without loss of the sperms' ability to fertilize. This approach has tremendous benefits for men who have difficulty producing sperm and for those with low and declining sperm counts.
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TL;DR: Possible correctable measures include foremost lifestyle changes, but also supplementation with antioxidants to scavenge excessive ROS, but this should only be done after careful examination of the patient and establishment of the individual bodily antioxidant needs.
Abstract: On the one hand, reactive oxygen species (ROS) are mandatory mediators for essential cellular functions including the function of germ cells (oocytes and spermatozoa) and thereby the fertilization process. However, the exposure of these cells to excessive levels of oxidative stress by too high levels of ROS or too low levels of antioxidative protection will render these cells dysfunctional thereby failing the fertilization process and causing couples to be infertile. Numerous causes are responsible for the delicate bodily redox system being out of balance and causing disease and infertility. Many of these causes are modifiable such as lifestyle factors like obesity, poor nutrition, heat stress, smoking, or alcohol abuse. Possible correctable measures include foremost lifestyle changes, but also supplementation with antioxidants to scavenge excessive ROS. However, this should only be done after careful examination of the patient and establishment of the individual bodily antioxidant needs. In addition, other corrective measures include sperm separation for assisted reproductive techniques. However, these techniques have to be carried out very carefully as they, if applied wrongly, bear risks of generating ROS damaging the germ cells and preventing fertilization.
87 citations
TL;DR: Frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI.
74 citations
Cites background from "Evaluation of human sperm function ..."
...While refrozen human spermatozoa have been assessed as to their ability to form pronuclei after injection into hamster oocytes [31], to our knowledge this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species....
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TL;DR: The results indicate that freezing stallion semen by MTG is superior to CRCM, and the differences according to all the evaluation methods were highly significant.
Abstract: Despite its potential impact on the horse industry, sperm cryopreservation is not an established technology throughout the industry, for a number of reasons that include a reduction in pregnancy rate and increased cost per pregnancy. We have evaluated a novel directional freezing technique, based on a multi-thermal gradient (MTG), by comparing it with the conventional, controlled-rate cryopreservation method (CRCM). Ninety-seven ejaculates with > or =50% motility, collected from 31 stallions were each divided into two parts and subsequently frozen by either MTG or CRCM. Frozen samples were then stored in liquid nitrogen until thawing. The two treatments were evaluated by three methods: progressive linear motility (PLM), viability stain and hypoosmotic swelling (HOS) test. High correlation was found between the three evaluation methods for all post-thaw samples. Eighty-eight per cent of the ejaculates frozen by MTG had post-thaw PLM > or =35%, whereas only 59% of the ejaculates frozen by CRCM had such motility. Post-thaw evaluations of samples frozen by MTG and CRCM were: PLM - 50.2 +/- 1.5% and 37.4 +/- 1.5%, respectively; viability - 53.6 +/- 1.5% and 39.5 +/- 1.4%, respectively; membrane integrity, as evaluated by HOS - 36.2 +/- 1.3% and 26.5 +/- 1.1%, respectively. The differences according to all the evaluation methods were highly significant (p < 0.001), and the results indicate that freezing stallion semen by MTG is superior to CRCM.
49 citations
TL;DR: Vitrification is superior to conventional freezing methods in preservation of spermatozoa, regarding total and progressive motility and the efficacy of vitrification is influence by using different vitrification protocol and cryopreservation of different quality spermatozosa, according to the results of meta-analysis.
45 citations
TL;DR: Both motility and vitality decreased steadily following each cycle but cell survival was significantly greater in the unwashed samples; however, samples that were not washed and to which fresh cryoprotectant was not added after each cycle fared significantly better than their washed counterparts.
34 citations
References
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TL;DR: Intracytoplasmic sperm injection is used to treat couples with infertility because of severely impaired sperm characteristics, and in whom IVF and SUZI had failed.
3,224 citations
"Evaluation of human sperm function ..." refers background in this paper
...The advent of intracytoplasmic sperm injection (ICSI) has shown that a single human sperm can be microinjected into an oocyte, bring about fertilization and embryogenesis, and when transferred to the uterus, results in the birth of a baby (Palermo et al, 1992)....
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TL;DR: It is demonstrated that with damaged spermatozoa, the key point in the fertilization process is the activation of the oocyte by injection of cytosolic sperm factor, and a similar fertilization rate can be achieved following activation.
Abstract: We report the first detailed and systematic study in a mammalian system to unravel the mystery of the beginnings of life. The fertilizing ability of damaged spermatozoa at various levels of disintegration (cellular and molecular) has been investigated in homologous (mouse) and heterologous (human spermatozoon, hamster oocyte) models. Live pups were produced after destruction of spermatozoa at various cellular and molecular levels followed by injection into oocytes. We demonstrate that with damaged spermatozoa, the key point in the fertilization process is the activation of the oocyte by injection of cytosolic sperm factor. A similar fertilization rate as that using live intact spermatozoa can be achieved following activation. However, the integrity of the genetic material influenced in-vitro development of the embryos and live fetuses. This study contributes to a better understanding of the fertilizing ability of damaged spermatozoa. These findings can be applied clinically to patients with necrozoospermia or very severe oligozoospermia and in wildlife research where damaged spermatozoa from rare species can be used to regenerate young, and hence propagate the species. Also implied is the possible contribution of sperm DNA strand breakage to early pregnancy loss.
182 citations
"Evaluation of human sperm function ..." refers background in this paper
...It was recently shown that mouse sperm that become nonviable through repeated freezing and thawing can fertilize mouse oocytes, with subsequent embryonic development following ICSI (Ahmadi and Ng, 1999)....
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TL;DR: In this paper, the ability of hypo-osmotic swelling test to select viable sperm from nonmotile sperm samples for intracytoplasmic sperm injection (ICSI) was evaluated.
157 citations
TL;DR: The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa in human, mouse or hamster zonaepellucidae.
Abstract: 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.
141 citations
"Evaluation of human sperm function ..." refers methods in this paper
...Insertion of sperm into empty zonae was carried out either by conventional microinjection (Cohen et al, 1997) or with the use of a laser beam (Montag et al, 1999)....
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TL;DR: In this paper, the effects of human seminal plasma on the fertilizing capacity of human spermatozoa were investigated using zona-free hamster eggs and salt-stored human eggs.
131 citations