Laboratory and Clinical Outcomes of Single Sperm Cryopreservation in Patients Underwent Micro-TESE

For men with severe oligozoospermia, sperm cryopreservation can preserve surgically obtained sperm. How to cryopreserve single sperm in men is still a hot topic in assisted reproduction technology. Aim to analyze the laboratory and pregnancy outcomes of single sperm cryopreservation group, we retrospectively selected 38 cycles underwent single sperm cryopreservation and thawing as the study group and 618 cycles underwent conventional sperm cryopreservation and thawing as the control group, which were performed in the reproductive medicine center of the Sixth Affiliated Hospital, Sun Yatsen University, from April 2014 to October 2023. All the sperm came from microdissection testicular sperm extraction (micro-TESE), and performed intracytoplasmic sperm injection (ICSI) for fertilization. Zygotes were cultured to Day 3 embryo, which were freshly transferred to female uterus. Surplus embryos were cultured to blastosphere and cryopreserved. There was no statistical difference in female/male age, female BMI, infertility duration and female basal sex hormone (FSH, LH E2, AMH), No. of oocytes retrieved per cycle, No. of ICSI oocytes per cycle and No. of embryos transferred per cycle between the two groups (P > 0.05). No significant difference was found in two-pronuclear oocyte fertilization rate (59.23% VS 58.84%), Day 3 available embryo rate (61.81% VS 63.55%), Day 3 good-quality embryo rate (45.73% VS 50.27%), blastocyst formation rate (47.83% VS 49.46%), the implantation rate (47.37% VS 52.16%), clinical pregnancy rate (36.84% VS 47.18%), miscarriage rate (14.29% VS 12.68%) and live birth rate (85.71% VS 81.70%) between two groups (P > 0.05). In conclusion, single-sperm cryopreservation was the optimal method to preserve sperm after micro-TESE. It can increase the utilization of each sperm and lead to clinical pregnancy.

The role of low-density lipoprotein （LDL） and high-density lipoprotein （HDL） in comparison with whole egg yolk for sperm cryopreservation in rhesus monkeys

Low-density lipoprotein （LDL） extracted from hen egg yolk has recently been considered to be superior to whole egg yolk in sperm cryopreservation of various animal species. Meanwhile, there was a notion that high-density lipoprotein （HDL） in egg yolk may have a negative effect on post-thaw survival. The role of LDL and HDL in sperm cryopreservation of rhesus monkeys has not been explored. The present study evaluates their effect in comparison with egg yolk with or without the addition of permeable cryoprotectant （glycerol） on sperm cryopreservation of rhesus macaques. In addition, various additives intended to change the lipid composition of LDL-sperm membrane complex have also been tested for their effectiveness in preserving post-thaw viability. Our findings indicated that LDL is the main component in egg yolk that is responsible for its protective role for sperm cryopreservation in rhesus monkeys. Regardless of the presence or absence of glycerol, the protective role of LDL is similar to that of egg yolk and we did not observe any superiority in post-thaw survival with LDL when compared to egg yolk. Modifying the lipid composition of LDL-sperm membrane complex with the addition of cholesterol, cholesterol loaded cyclodextrin and phosphatidylcholine also did not yield any improvements in pest-thaw survival; while addition of methyl-β-cyclodextrin reduced post-thaw motility. HDL plays a neutral role in sperm cryopreservation of rhesus monkeys. The present study suggests that egg yolk may still hold advantages when compared with LDL as effective components in extenders for sperm cryopreservation in rhesus monkeys.

Preservation of sperm of cancer patients： extent of use and pregnancy outcome in a tertiary infertility center

Sperm cryopreservation is the best modality to ensure future fertility for males diagnosed with cancer. The extent to which cryopreserved sperm is actually used for impregnation, the fertility treatment options that are available and the success rates of these treatments have not been investigated in depth. The medical records of 682 patients who cryopreserved sperm cells due to cancer treatment were analyzed. Seventy of these patients withdrew their frozen sperm for fertility treatments over a 20-year period （most within the first 4 years after cryopreservation）. Sperm quality of different malignancies and outcomes of assisted reproduction treatment （ART） for pregnancy achievement in relation to the type of treatment and the type of malignancy were evaluated. The results showed that the rate of using cryo-thawed sperm from cancer patients for fertility treatments in our unit was 10.3%. Sperm quality indices differed between different types of malignancies, with the poorest quality measured in testicular cancer. Conception was achieved in 46 of the 184 ART cycles （25%）, and resulted in 36 deliveries. The use of intracytoplasmic sperm injection （ICSI） methodology yielded a significantly higher pregnancy rate （37.4%） than intrauterine insemination （IUI; 11.5%） and was similar to other groups of infertile couples using these modalities. In vitro fertilization （IVF） failed to produce pregnancies. In conclusion, the rate of use of cryopresseved sperm in cancer patients is relatively low （10.3%）. Achievement of pregnancies by ICSI presents the best option but when there are enough stored sperm samples and adequate quality, I UI can be employed. Cryopreservation is nevertheless the best option to preserve future fertility potential and hope for cancer patients.

Stem cell-based therapies for fertility preservation in males:Current status and future prospects

With the decline in male fertility in recent years,strategies for male fertility preservation have received increasing attention.In this study,by reviewing current treatments and recent publications,we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation,focusing on the use of spermatogonial stem cells(SSCs),SSC niches,SSC-based testicular organoids,other stem cell types such as mesenchymal stem cells,and stem cell-derived extracellular vesicles.In conclusion,a more comprehensive understanding of the germ cell microenvironment,stem cell-derived extracellular vesicles,and testicular organoids will play an important role in achieving male fertility preservation.

A retrospective study on sperm cryopreservation in 1034 patients diagnosed with cancer in southern China

Sperm cryopreservation is an effective fertility preservation method for cancer patients before anticancer treatments. However, thereare little data on fertility preservation in large cohorts of patients with cancer in southern China. This retrospective cross-sectionalstudy aimed to assess the fertility preservation status of 1034 newly diagnosed male patients with cancer in the Human SpermBank of Guangdong Province in southern China (Guangzhou, China). Of these, 302 patients had reproductive system tumors,mostly testicular cancers (99.0%), and 732 had other tumors, including lymphoma (33.1%), gastrointestinal cancer (16.3%),nasopharyngeal carcinoma (15.7%), leukemia (7.7%), sarcoma (3.6%), and others (23.6%). Patients with reproductive systemtumors had lower sperm concentration and prefreezing and post-thawing progressive motility than those with non-reproductive systemtumors (all P < 0.001). Differences in sperm concentration, progressive motility, and normal morphology rate were observed betweenpatients with and without anticancer surgery before sperm cryopreservation (all P < 0.05). As of April 30, 2022, 63 patients usedtheir cryopreserved sperm for assisted reproductive technology treatments and 39 pregnancies were achieved. This study providesvaluable data on the fertility preservation status in newly diagnosed cancer patients in southern China, demonstrating that patientswith reproductive system tumors had poor sperm quality for their pretreatment fertility preservation.

Update on techniques for cryopreservation of human spermatozoa

In the 1960s,sperm cryopreservation was developed as a method to preserve fertility.Currently,techniques for the cryopreservation of human spermatozoa have been widely used in assisted reproduction.However,although sperm cryobiology has made notable achievements,the optimal method for the recovery of viable spermatozoa after cryopreservation remains elusive.Postthawing sperm quality can be affected by cryoprotectants,ice formation,storage conditions,and osmotic stress during the freezing process.This review discusses recent advances in different cryopreservation techniques,cryoprotectants,and freezing and thawing methods during cryopreservation and new indications for the use of cryopreserved spermatozoa.

Green tea extract as a cryoprotectant additive to preserve the motility and DNA integrity of human spermatozoa

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_(2)O_(2)).Spermatozoa were treated with GTE at different concentrations before incubation with H_(2)O_(2).In part two,the semen of 45 patients was cryopreserved with or without 1.0 ng ml^(-1)GTE.After 2 weeks,the semen was thawed,and the effect on sperm motility and DNA fragmentation was observed.Our data showed that GTE significantly protected sperm motility and DNA integrity against oxidative stress induced by H_(2)O_(2)when added at a final concentration of 1.0 ng ml^(-1).We found that the addition of 1.0 ng ml^(-1)GTE to cryopreservation media significantly increased sperm motility and DNA integrity(both P<0.05).More interestingly,patients with high sperm DNA damage benefited similarly from the GTE supplementation.However,there was no significant change in the reactive oxygen species(ROS)level.In conclusion,supplementing sperm freezing media with GTE has a significant protective effect on human sperm motility and DNA integrity,which may be of clinical interest.

Clinical benefits of a modified Cryopiece system for cryopreservation of rare ejaculated and testicular spermatozoa for ICSI

Cryopreservation of rare testicular-retrieved spermatozoa for intracytoplasmic sperm injection(ICSI)in patients with severe oligozoospermia and azoospermia remains a major challenge in clinical practice.This study evaluated the Cryopiece system as a potential technique to cryopreserve rare human spermatozoa for ICSI.Small numbers of ejaculated(24 patients)and testicular(13 patients)spermatozoa were cryopreserved using the Cryopiece system.The total number of recovered spermatozoa and motility were assessed after thawing.Thirty-seven couples underwent ICSI using spermatozoa cryopreserved by the Cryopiece system,and ICSI outcomes(rates of fertilization,embryo cleavage,and clinical pregnancy)were evaluated.The average sperm post-thaw retrieval rate was 79.1%,and motility was 29.7%.Ejaculated spermatozoa had a higher post-thaw motility(32.5%)than testicular spermatozoa(21.8%;P=0.005).ICSI achieved a fertilization rate of 61.9%,embryo cleavage rate of 84.6%,and clinical pregnancy rate of 43.3%.The ICSI outcomes in the ejaculated and testicular frozen-thawed spermatozoa were similar.Assisted oocyte activation(AOA)after ICSI with motile(72.1%)or immotile(71.9%)spermatozoa resulted in a significantly higher fertilization rate than that when using motile spermatozoa without AOA(52.0%;P=0.005).However,AOA did not enhance the clinical pregnancy rate(55.6%or 40.0%vs 35.3%;P=0.703).The Cryopiece system is simple and useful for the cryopreservation of small numbers of ejaculated or testicular spermatozoa for ICSI in patients with severe oligozoospermia or nonobstructive azoospermia.