Comparison of the Effects of Single Sperm Cryopreservation and Conventional Cryopreservation on Embryo Culture and Pregnancy in Patients Undergoing Micro-TESE / 中山大学学报(医学科学版)

ObjectiveTo compare the effects of single sperm cryopreservation and conventional cryopreservation on embryo culture and pregnancy in patients undergoing microdissection testicular sperm extraction （micro-TESE）. MethodsA retrospective analysis was done on the patients who underwent micro-TESE at the Reproductive Medicine Center in the Sixth Affiliated Hospital of Sun Yat-Sen University between January 2018 and December 2021. The single sperm cryopreservation group included 39 patients undergoing single sperm cryopreservation and 307 MII oocytes. The conventional cryopreservation group included 39 patients undergoing conventional cryopreservation and 410 MII oocytes. Propensity score matching （PSM） was performed to balance the selection bias. The fertility rate， embryo culture and pregnancy of these two groups were compared. ResultsThere was no statistical difference in age， body mass index （BMI）， years of infertility， basal FSH， basal LH， basal E2， AMH， AFC， number of oocytes retrieved and number of transferred embryos between the two groups （P>0.05）. No significant difference was found in fertilization rate （65.8% vs. 60.49%）， available embryo rate （67.82% vs. 58.87%） and high-quality embryo rate （70.80% vs. 75.34%）. The single sperm cryopreservation group had significantly lower clinical pregnancy rate than conventional cryopreservation group （45.45% vs. 70.0%， P=0.049）. ConclusionIf the patients undergoing micro-TESE have very few sperms， single sperm cryopreservation could be an effective choice to increase the utilization of each sperm and ensure the subsequent sperm retrieval after thawing， but the clinical pregnancy rate is decreased. Due to the limited number of cases， we need a large additional number of cases to observe and analyze.

Green tea extract as a cryoprotectant additive to preserve the motility and DNA integrity of human spermatozoa / 亚洲男科学杂志(英文版)

Cryopreservation impairs sperm quality and functions, including motility and DNA integrity. Antioxidant additives in sperm freezing media have previously brought improvements in postthawed sperm quality. Green tea extract (GTE) is widely considered as an excellent antioxidant, and its beneficial role has been proven in other human cells. This study aims to evaluate the GTE as a potential additive in cryopreservation media of human spermatozoa. In part one, the semen of 20 normozoospermic men was used to optimize the concentration of GTE that maintains sperm motility and DNA integrity against oxidative stress, induced by hydrogen peroxide (H

Increased cryo-survival rate in ejaculated human sperm from infertile men following pre-freeze in vitro myo-inositol supplementation / 대한생식의학회지

OBJECTIVE: To investigate the effects of in vitro myo-inositol (Myo-Ins) supplementation of cryopreserved human semen on the cryo-survival rate (CSR). METHODS: Semen samples were obtained from 41 infertile men. Following routine semen analysis, each sample was divided into two equal aliquots (0.5 mL each). One aliquot was treated with 1 mg of Myo-Ins dissolved in 10 µL of sperm preparation medium. The second aliquot was treated with 10 µL of the same medium (control). Both aliquots were incubated for 20 minutes prior to freezing to slow the freezing process. The frozen samples were examined for post-thaw percentages of total motility (TM), progressive motility (PM), and the CSR, defined as the percentage of post-thaw TM divided by the percentage of pre-freeze TM and multiplied in 100. The results were expressed as median and interquartile range (25th and 75th percentiles). RESULTS: The pre-freeze TM (50% [30%–50%]) and PM (35% [20%–35%]) were significantly higher than the post-thaw TM and PM in the Myo-Ins group (15% [10%–35%] and 10% [5%–20%]; p < 0.001 and p < 0.001, respectively) and the control group (10% [6%–30%] and 5% [3%–15%]; p < 0.001 and p < 0.001, respectively). The CSR of the 41 semen aliquots supplemented with Myo-Ins (40% [25%–70%]) was significantly higher than that of the control samples (30% [13%–58%], p=0.041). The CSR of the 26 abnormal semen samples that were supplemented with Myo-Ins (38% [20%–50%]) was significantly higher than that of the control samples (23% [12%–30%], p=0.031). CONCLUSION: In vitro Myo-Ins supplementation of ejaculated human sperm from infertile men resulted in a significant increase in the CSR in samples with abnormal pre-freeze sperm parameters.

Current progress in single sperm cryopreservation / 中华男科学杂志

Sperm cryopreservation has been widely used in assisted reproduction, but conventional techniques are not suitable for the cryopreservation of small numbers of sperm. The application of the single sperm cryopreservation technique has significantly improved the clinical treatment of cryptozoospermia and non-obstructive azoospermia. Ever since Cohen et al first developed the method of single sperm cryopreservation in 1997, constant efforts have been made to develop the carriers for this technique. In this review, we mainly discuss the existing methods and clinical outcomes of single sperm cryopreservation.

Plasminogen activator inhibitor 1 and Antipain preserve acrosome integrity of bovine spermatozoa during cryopreservation / Inibidor do ativador do plasminogênio 1 e antipaína preservam a integridade do acrossoma de espermatozoides bovinos durante a criopreservação

Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.(AU)

A criopreservação é parcialmente prejudicial à fertilidade do sêmen de bovinos e induz mudanças semelhantes à capacitação em espermatozoides. O plasma seminal contém serina-proteases e inibidores de serina-proteases que estão envolvidos na fertilização de mamíferos, e os inibidores podem ser aplicados para evitar uma capacitação espermática induzida pelo frio. Analisaram-se os efeitos de diferentes concentrações de dois inibidores de serina-proteases, inibidor do ativador do plasminogênio 1 - PAI-1 (70ƞg, 140ƞg e 210ƞg) e antipaína (10µg, 50µg e 100µg) na suplementação ao diluidor de criopreservação de sêmen bovino. Trinta e seis ejaculados de quatro bovinos Curraleiro Pé-Duro foram usados para criopreservação. Os efeitos dos inibidores sobre os parâmetros dos espermatozoides (cinética espermática - CASA, integridade acrossomal, integridade da membrana plasmática, potencial de membrana mitocondrial, defeitos espermáticos e taxa de reação acrossomal) foram avaliados no sêmen pós-descongelamento. A criopreservação de espermatozoides com antipaína diminuiu os parâmetros cinéticos pós-descongelamento de MP, VSL, LIN, SRT e a porcentagem de espermatozoides hiperativados, PAI-1 (210ƞg) diminuiu VSL e LIN. Antipaína e PAI-1 não tiveram efeitos nos parâmetros de integridade da membrana plasmática, no potencial de membrana mitocondrial e nos defeitos espermáticos. Espermatozoides criopreservados na presença de antipaína e PAI-1 (70 e 140ƞg) preservaram a integridade acrossomal, assim como foram capazes de completar a reação acrossômica in vitro. Em conclusão, os inibidores de serina-proteases, antipaína e PAI-1 (70 e 140ƞg) são capazes de preservar a integridade acrossomal de espermatozoides criopreservados de bovinos.(AU)

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs / Teste de ligação espermática à membrana perivitelínica da gema de ovo de galinha como avaliação funcional do sêmen de cães

During fertilization, spermatozoa interact with the zona pellucida (ZP) through the binding between the acrosome and proteins 2 and 3 (ZP2 and ZP3). The perivitelline membrane of chicken egg yolk is homologous to the mammalian ZP3, which allows the binding of sperm of several species. The aim of this study was to standardize and evaluate the efficiency of sperm-binding to the perivitelline membrane of chicken eggs as a functional method for canine semen evaluation. For this purpose, nine post-thaw sperm samples were used, which were divided into two aliquots: the first was kept in water bath at 37ºC (live sample) and the second was submitted to cold shock to induce cellular damage (dead sample). The two aliquots were mixed on five proportions, corresponding to 0%, 25%, 50%, 75%, and 100% of viable cells, and the binding test was performed by analyzing the number of spermatozoa bonded to the perivitelline membrane by means of computerized assessment of sperm motility (CASA) or conventional microscopy. Additionally, samples were submitted to sperm motility analysis, evaluation of plasmatic and acrosomal membrane integrity, and sperm mitochondrial activity. The sperm-binding test to the perivitelline membrane of chicken egg yolk was considered a feasible sperm analysis test for both fertilizing capacity and overall sperm attributes evaluation, mainly when the analysis is performed by a conventional microscope, which expands its practicality to the majority of canine reproduction laboratories.(AU)

Durante a fecundação, os espermatozoides interagem com a zona pelúcida (ZP) por meio da ligação entre o acrossomo e as proteínas 2 e 3 (ZP2 e ZP3). A membrana perivitelínica da gema de ovo de galinhas é homóloga à ZP3 de mamíferos, possibilitando a ligação espermática de diversas espécies. Este trabalho padronizou e avaliou a eficiência do teste de ligação espermática à membrana perivitelínica da gema de ovo de galinhas como avaliação funcional do sêmen de cães. Para tal, foram utilizadas nove amostras seminais previamente criopreservadas. Cada amostra foi dividida em duas alíquotas: a primeira foi mantida em banho-maria à 37ºC (vivos) e a segunda submetida a choque térmico com o intuito de induzir dano celular (mortos). As duas alíquotas foram misturadas, correspondendo a 0, 25, 50, 75 e 100% de células viáveis. As amostras foram avaliadas quanto ao número de espermatozoides ligados à membrana perivitelínica por meio da análise computadorizada da motilidade (CASA) ou microscopia convencional. Ademais, as amostras foram avaliadas quanto à motilidade espermática, integridade das membranas acrossomal e plasmática e atividade mitocondrial espermática. O teste de ligação espermática à membrana perivitelínica de ovos de galinha foi considerado um teste de análise seminal exequível tanto para avaliar a capacidade fecundante dos espermatozoides como atributos seminais gerais, especialmente quando realizado em microscopia convencional, expandindo sua praticidade para a maioria dos laboratórios de análise de sêmen canino.(AU)

Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes of assisted conception

The introduction of the technique of intracytoplasmic sperm injection to achieve fertilization, especially using surgically retrieved testicular or epididymal sperm from men with obstructive or non-obstructive azoospermia, has revolutionized the field of assisted reproduction. The techniques for the retrieval of spermatozoa vary from relatively simple percutaneous sperm aspiration to open excision (testicular biopsy) and the more invasive Micro-TESE. The probability of retrieving spermatozoa can be as high as 100% in men with obstructive azoospermia (congenital bilateral absence of the vas deferens, status post-vasectomy). However, in nonobstructive azoospermia, successful sperm retrieval has been reported in 10-100% of cases by various investigators. The surgical retrieval and cryopreservation of sperm, especially in men with non-obstructive azoospermia, to some extent ensures the availability of sperm at the time of intracytoplasmic sperm injection. In addition, this strategy can avoid unnecessary ovarian stimulation in those patients intending to undergo in vitro fertilization-intracytoplasmic sperm injection with freshly retrieved testicular sperm when an absolute absence of sperm in the testis is identified. Several different methods for the cryopreservation of testicular and epididymal sperm are available. The choice of the container or carrier may be an important consideration and should take into account the number or concentration of the sperm in the final preparation. When the number of sperm in a testicular biopsy sample is extremely low (e.g., 1-20 total sperm available), the use of an evacuated zona pellucida to store the cryopreserved sperm has been shown to be an effective approach.

Qualitative assessment of washed human normal sperm after cryopreservation

Background: Sperm cryopreservation becomes a relatively routine process in assisted reproductive centers. However, there must be ensured quality of washed human normal sperm and cryopreservation to successful fertilization. Objective: To evaluate the quality of washed human normal sperm after cryopreservation. Subjects and method: 30 normal semen samples, each sample was divided into two parts for washed and unwashed spermatozoa. All samples were cryopreserved in 1, 2 and 30 days. Evaluating and comparing the quality of sperm before and after which washed, pre-cryopreservation and post-cryopreservation between the groups were performed. Results: The quality of sperm after washing was more significantly improved than before washing. Post-cryopreservation, the quality of sperm was reduced time by time but within an accepted limitation. There was not a significant difference between the two ways of preparation before cryopreservation. Conclusions: The quality of sperm at post-cryopreservation was reduced (both washed sperm and unwashed sperm). The quality of washed sperm is reduced continuously with time, but there was no difference between the two studied groups.

Effects of Cryopreservation on Morphological Characteristics of Washed Spermatozoa from Normozoospermia

Introduction: Successful cryopreservation of spermatozoa must ensure normal newborns after the preservation time. This method frequently can potentially contain cross-infected risks during the cryopreservation process in the liquid nitrogen environment (such as HCV, HIV). A number of researchers reveal that these risks can be eliminated by washing spermatozoa before cryopreservation. However, the problem is whether cryopreservation of washed spermatozoa still retains its morphology and function or not? \r\n', u'Objectives: To evaluate the change of sperm morphology characteristics after which washed sperm cryopreserved from normozoospermia. \r\n', u'Subjects and method: 30 normal semen samples; each sample was divided into two aliquots of washed and unwashed spermatozoa. All samples were cryopreserved in stages of 1, 2 and 30 days. We compared the percentage of spermatozoa with normal morphology before and after which was washed, pre - cryopreservation and post - cryopreservation between the groups. \r\n', u'Results: The percentage 0 spermatozoon with normal morphology after washing was more significantly increased than prior to washing. Post - cryopreservation, this percentage was reduced time by time but acceptable. There is no significant difference between the two ways of preparation before cryopreservation. The percentage of spermatozoa with abnormal head and neck increased significantly after cryopreservation. \r\n', u'Conclusion: The percentage of spermatozoa with normal morphology post - cryopreservation was reduced in both washed sperm and unwashed sperm samples. This percentage was reduced time by time, but there is no difference between the two groups studied. \r\n', u'\r\n', u'

Comparison between Isolate(R) gradient and Swim-up Procedures for Sperm Preparation: Effects on Freeze-thawing in Normal Semen Sample / 대한불임학회지

No abstract available in English.

Effects of Anti-oxidants in Freezing and Thawing of Sperm / 대한비뇨기과학회지

PURPOSE: It is known that reactive oxygen species which increase during the cryopreservation of sperm are harmful to sperm. This study analyzed the concentration, motility, morphology and lipid peroxidation of cell membrane before and after the cryopreservation of sperm for an evaluation of the influences of reactive oxygen species in sperm function. MATERIALS AND METHODS: Semen samples from 50 normal healthy volunteers were collected by masturbation. After liquefaction at room temperature, the semen was divided and stored in cryogenic tubes supplemented with Ham`s F-10 medium and a cryoprotective agent. To the experimental group was added superoxide dismutase(SOD, Sigma, USA) 100microliter, catalase (Sigma, USA) 100microliter, SOD and catalase 50microliter and 200microliter, glutathione peroxidase 10microliter, SOD and glutathione peroxidase 50microliterand 20microliter. It was then divided into groups I, II, III, IV and V. Antioxidant was not added to the control group. All specimens were cryopreservated at -196degrees C liquid nitrogen, then thawed at room temperature. We analyzed sperm motility and morphology, and the level of lipid peroxidation was measured by thiobarbituric acid(TBA). RESULTS: The sperm concentration and morphology did not show any significant differences statistically between the experimental and the control groups. After cryopreservation, the sperm motility was significantly decreased in the experimental and the control groups(p<0.05). In groups II, III, IV and V, the sperm motility decreased more than in group I and the control group(p<0.05). In groups II, III, IV and V, lipid peroxidation of cell membrane was significantly lower than in group I and the control group(p<0.05). In both the experimental and the control groups, the ratio of motile sperm decreased significantly, according to increasing lipid peroxidation(p<0.05, r=0.566). CONCLUSIONS: We conclude from this study that the reactive oxygen species occuring in sperm cryopreservation may significantly influence the function of sperm. A cryoprotective agent supplemented with proper anti-oxidant may reduce the harmful effect associated with sperm cryopreservation.