Search

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. [ABSTRACT FROM AUTHOR]

Background: Systematic reviews have focused on sperm recovery and post‐thaw parameters after cryopreservation, but there is no information on the associated clinical outcomes. In recent years, an increasing number of studies have reported cryopreservation of a single sperm due to the importance of fertility preservation. Objectives: To assess whether the cryopreservation of single human spermatozoa improves clinical outcomes in patients with azoospermia or severe oligospermia. Materials and methods: We conducted an extensive literature search using the following databases: CENTRAL, CNKI, Cochrane Systematic Reviews, EMBASE, MEDLINE, PUBMED, and Web of Science for relevant studies published through December 31, 2019. We calculated the pooled proportions of cryopreservation of a single human spermatozoon to assess the recovery, survival, fertilization, pregnancy, miscarriage, and delivery rates. Subgroup analyses were performed for the following covariates, (a) different carriers, (b) year of publication, and (c) source of sperm. Results: We included 25 studies, which included 13 carriers. The pooled proportion of recovery rate of spermatozoa cryopreserved was 92% (95% CI, 87%–96%), and the survival, fertilization, pregnancy, miscarriage, and delivery rates were 76% (95% CI, 69%–83%), 63% (95% CI, 58%–67%), 57% (95% CI, 39%–74%), 12% (95% CI, 0%–33%), and 40% (95% CI, 12%–71%), respectively. Based on the subgroup analysis, the recovery and survival rates of frozen spermatozoa in a subgroup of different carriers were statistically significant. In the past decade, frozen single human spermatozoon technology has improved the recovery rates of frozen‐thawed spermatozoa. However, the differences in clinical outcomes of frozen spermatozoa in subgroups of different sources of sperm were not statistically significant. Discussion and conclusion: The techniques for single human spermatozoa are feasible and efficient and may benefit patients with severe oligospermia or azoospermia. [ABSTRACT FROM AUTHOR]

Sperm morphology was once believed as one of the most predictive indicators of pregnancy outcome in assisted reproductive technology (ART). However, the impact of teratozoospermia on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcomes and its offspring remains inconclusive. In order to evaluate the influence of teratozoospermia on pregnancy outcome and newborn status after IVF and ICSI, a retrospective study was conducted. This was a matched case-control study that included 2202 IVF cycles and 2574 ICSI cycles and was conducted at the Reproductive and Genetic Hospital of CITIC-Xiangya in Changsha, China, from June 2013 to June 2018. Patients were divided into two groups based on sperm morphology: teratozoospermia and normal sperm group. The pregnancy outcome and newborn outcome were analyzed. The results indicated that couples with teratozoospermia had a significantly lower optimal embryo rate compared to those with normal sperm morphology in IVF (P = 0.007), while there were no statistically significant differences between the two groups in terms of the fertilization rate, cleavage rate, implantation rate, and pregnancy rate (all P > 0.05). Additionally, teratozoospermia was associated with lower infant birth weight in multiple births after IVF. With regard to ICSI, there was no significant difference in both pregnancy outcome and newborn outcome between the teratozoospermia and normal groups (both P > 0.05). Furthermore, no increase in the risk of birth defects occurred in the teratozoospermia group after IVF/ICSI. Consequently, we believe that teratozoospermia has limited predictive value for pregnancy outcomes in IVF/ICSI, and has little impact on the resulting offspring if multiple pregnancy is avoided. [ABSTRACT FROM AUTHOR]

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice. However, it has been shown to have a negative impact on sperm function and structure. Vitrification as a successful alternative method has been proved to have better protective effects on human embryos, but vitrification of spermatozoa is still subject to low recovery rates. In this study, a modified vitrification method for native spermatozoa was developed. A total of 28 semen samples were included; each sample was divided into three equal parts and assigned to fresh, slow freezing, and vitrification groups. Sperm vitality, motility, morphology, DNA integrity, and acrosome reaction were assessed for each of the groups. The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing; vitrification achieves a higher recovery rate (P < 0.05), motility (P <0.05), morphology (P <0.05), and curve line velocity (P <0.05) than slow freezing. Furthermore, DNA fragmentation was decreased (P <0.05) and better acrosome protection (P <0.05) was exhibited in the spermatozoa after vitrification. Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster, indicating that spermatozoa are better preserved through vitrification. In conclusion, while both slow freezing and vitrification have negative effects on sperm function and structure, the vitrification protocol described here had a relatively better recovery rate (65.8%) and showed improved preservation of several sperm quality parameters compared with slow freezing. [ABSTRACT FROM AUTHOR]

Pericentric inversion of chromosome 9 (inv[9]) is a common chromosomal structural variant, but its impact on clinical outcomes remains debated. The screening criteria of sperm banks are rarely mentioned to individuals with inv(9). In this study, we evaluated the fertility of sperm donors with inv(9) who met eligibility criteria for sperm banks (inv[9]-eligible donors). From March 2004 to May 2022, chromosomal analysis of 16 124 sperm donors at CITIC-Xiangya Human Sperm Bank in Hunan Province (Changsha, China) found that 251 (1.6%) had chromosome variations, with inv(9) being the most prevalent at 1.1%. All 169 inv(9)-eligible donors were contacted to collect fertility outcome data, along with 206 eligible donors without inv(9) as controls. In addition, semen samples from inv(9)-eligible donors and eligible donors underwent assessments of sperm fluorescence in situ hybridization (FISH), mitochondrial membrane potential, DNA fragmentation index, acrosome integrity, reactive oxygen species (ROS), and sperm morphology. Results showed that inv(9) did not significantly increase reproductive risks overall. Despite detecting ROS level differences, the clinical impact may be insignificant. This study provides new data on the inv(9) population that can serve as a valuable reference for decision-making by sperm banks as well as for genetic counseling and clinical guidance for individuals carrying inv(9) variant., (Copyright © 2024 Copyright: © The Author(s)(2024).)

The global practice of cryopreservation of human semen is commonplace in Assisted Reproductive Technology (ART) labs and sperm banks. However, information on the effects of long-term cryopreservation on semen is limited to clinical data summaries and descriptions. For this study, we prepared 4 semen specimens of fresh semen, 4 specimens cryostored for at least 1 year, 3 specimens cryostored for at least 5 years, 4 specimens cryostored for at least 10 years, and 3 specimens cryostored for at least 15 years. Total RNA was extracted from each sample, amplified, labeled, and mapped to the known primary microRNA (miRNA) in the miRBase database, enabling the prediction of novel miRNAs. We found that cryopreservation can lead to changes in miRNA expression, and with the increase in storage time, these changes became more pronounced. Meanwhile, the expression of let-7d-3p, let-7c-5p and let-7i-3p miRNAs changed dynamically over cryostorage time in frozen-thawed human sperm. Furthermore, we analyzed the time-dependent dynamics of cryostorage-expressed miRNAs and their target mRNAs and found that half of the target genes were expressed in oocytes. These intersection genes were mainly enriched in cancer and cytoskeletal signaling pathways. Our findings showed that the miRNA expression profile of cryopreserved human semen is modified by long-term storage. Furthermore, as the storage time increases, the impact on human sperm becomes more pronounced in terms of miRNAs, which may have an effect on subsequent fertilization and embryonic development.

Recent studies have confirmed that, in addition to sperm DNA, environmental exposure factors such as parental diet structure and stress state affect early embryonic development and offspring growth, thus leading to cross-generational inheritance of acquired traits. Many studies also show that environmental stressors can change the expression level of sperm small non-coding RNAs (sncRNAs). Furthermore, as the carrier of paternal genetic information transmission and epigenetic marker, sncRNAs are directly or indirectly involved in epigenetic regulation, mediating inter-generational inheritance of acquired traits. This review focuses on the two sncRNAs derived from microRNA (miRNA) and tRNA (tsRNA) in sperm epigenetics research.

Infertility affects approximately 20% of childbearing couples in the world, and azoospermia accounts for 10－15% of the causes of male infertility. The use of fresh or frozen-thawed testicular sperm for intracytoplasmic sperm injection (ICSI) has become a main method for azoospermia patients to realize their dream for reproduction. However, testicular spermatozoa are not further matured in the epididymis and therefore have an obviously lower anti-freezing ability than ejaculated sperm. The viability and retrieval rate of sperm are low after freeze-thaw with the conventional method of cryopreservation. Since the first live birth with frozen-thawed testicular spermatozoa, continuous improvement has been made in the methods of testicular sperm cryopreservation and increased the viability and retrieval rate of spermatozoa after freeze-thaw. This review focuses on the methods of testicular sperm cryopreservation in the past 20 years to provide a theoretical basis for the development of assisted reproductive technology.

Objective: To explore the correlation of Mycoplasma genitalium (MG) infection with male infertility., Methods: Totally, 27 314 males with infertility and 200 fertile sperm donors underwent MG and routine seminal examinations. The infertile men were divided into azoospermia, oligozoospermia, asthenozoospermia, oligoasthenospermia, and normal semen quality groups based on the results of seminal examination, the 27 286 of them with age data into eight age groups (<21, 21－25, 26－30, 31－35, 36－40, 41－45, 46－50 and ≥51 years old), and the 9 058 with definite diagnosis into primary and secondary infertility groups. Fifty-six cases of MG infection among the infertile males were treated with antibiotics for 2 weeks and examined for changes of the semen parameters., Results: Compared with the normal controls, the oligozoospermia patients showed a significantly higher rate of MG infection (0.50% vs 3.62%, P = 0.024), the highest in the ≥51 yr group (3.68%, P = 0.021), followed by the 21－25 yr group (3.00%, P = 0.048), and so did the infertile males (3.64%, P = 0.011), the men with primary infertility (3.73%, P = 0.010) and those with secondary infertility (3.57%, P = 0.015). MG infection was found to be associated with oligozoospermia (OR = 7.471, 95% CI: 1.001－55.784), primary infertility (OR = 7.704, 95% CI: 1.073－55.309) and secondary infertility (OR = 7.362, 95% CI: 1.026－52.837) but not with the age of the patients. Both sperm concentration and sperm count were significantly lower in the infected men before treatment than in the non-infection group after treatment (P < 0.05)., Conclusions: MG infection is related to male infertility and reduces the semen volume and sperm concentration, but does not affect sperm motility.

Objective: To investigate the pregnancy outcomes of assisted reproductive technology (ART) with cryopreserved donor sperm and the safety of the offspring thus conceived., Methods: The Human Sperm Bank of CITIC Xiangya Hospital provided cryopreserved donor semen to 31 reproductive centers in China between January 2006 and December 2012, with which 50247 ART cycles were accomplished. We compared the rates of birth defects and spontaneous abortion of intracervical insemination (ICI), intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI)., Results: A total of 39 047 ART cycles were performed by artificial insemination with cryopreserved donor sperm, including 36 674 cycles of ICI and 2 372 cycles of IUI. Among the 8 612 clinical pregnancies achieved by ICI, there were 917 cases of spontaneous abortion (at <28 gestational wk) (10.6%) and 6133 live births, with 43 cases of birth defect (0.70%). Of the 547 clinical pregnancies achieved by IUI, there were 41 cases of spontaneous abortion (7.5%) and 426 live births, with 2 cases of birth defect (0.47%). Totally, 11 200 cycles of IVF and ICSI were accomplished with cryopreserved donor sperm. Of the 5 860 clinical pregnancies achieved by IVF, there were 456 cases of spontaneous abortion (7.8%) and 5089 live births, with 55 cases of birth defect (1.08%). Among the 350 clinical pregnancies achieved by ICSI, there were 30 cases of spontaneous abortion (8.6%) and 229 live births, with 3 cases of birth defect (1.31%). The birth defect rate of ART with cryopreserved donor sperm was significantly lower than that published by the Chinese Ministry of Health (0.86% vs 1.53%,P<0.01)., Conclusions: The safety of the offspring conceived by ART with cryopreserved donor sperm is controllable.

Objective: To establish a long-term proliferation culture system for mouse spermatogonial stem cells., Methods: Testis tissues were obtained from 30 newborn male ICR mice on postnatal day 2-6. Testis cell suspension was collected by two-step enzymatic digestion prior to culture. The dissociated cells were aliquoted into tissue culture plates and cultivated with a modified system composed of serum-free defined medium on mouse embryonic fibroblasts (MEF) feeders. Their proliferation was determined by the BrdU incorporation test and the cultured cells identified by alkaline phosphatase (AP) activity, immunofluorescence staining and RT-PCR assay., Results: The cultures remained in a steady state and continued to generate germ cell colonies. The undifferentiated state was confirmed by strong positivity for AP activity, immunofluorescent staining of GFRalpha-1+ /Oct-4+ /VASA+ /SCP3- and GFRalpha-1+ /Oct-4+/SCP3- at the gene expression levels., Conclusion: Mouse spermatogonial stem cells could be expanded in our defined culture system and passaged steadily in vitro. The harvested cells remained in an undifferentiated state, which has provided a good platform for the study of spermatogenesis in vitro.

Objective: To separate the low abundance protein and establish the 2-DE synthetic map of total protein of human normal spermatozoa by using the 2-DE technology., Methods: All the needed human spermatozoa were collected and mixed, and proteins were extracted at one time with the method of urea/thiourea and ultra-sound. 0.8 mg, 0.6 mg, 0.5 mg, 0.3 mg sperm protein extracts were separated with 2-DE. Analyzed with MALDI-TOF-MS, PI and MW of 2 spots were obtained. Then set the 2 spots as the referent spots, different maps were compared and analyzed. At last, a synthetic map enriched with low abundance protein was obtained., Results: 1,080 +/- 23 protein spots have been separated on the 2-DE map with standard 0.5 mg loading amount and a synthetic map A was constructed which consist of 889 matched protein spots on the all maps with 0.5 mg loading amount. 381, 50 and 32 new spots were detected individually on the maps with 0.8 mg, 0.6 mg and 0.3 mg protein loading amount. A synthetic map with 1,352 protein spots was obtained., Conclusion: Low abundance protein was separated and a synthetic map enriched with low abundant protein was obtained by changing the protein loading amount.

In this study, a new data mining tool called Digital Differential Display (DDD) from the NCBI was used to predict testis-specific expressed genes from the expressed sequence tag (EST) database. DDD (digital differential display) was performed between nine testis libraries and seventy libraries derived from other tissues. We identified a new contig of ESTs (HS. 326528) which was from testis libraries. To validate the use of bioinformatic approaches in gene discovery, the ESTs (HS. 326528), which were predicted to be testis-specific, were chosen for further study. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from testis and other tissues indicated that the ESTs were specifically expressed in testis. This result was further validated by multi-tissue Northern blot. The full-length cDNA encompassing the entire open reading frame was cloned and, in view of its apparent specificity to testis, the gene was termed homo sapiens spermatogenesis-related gene 8---SRG8 (GenBank accession number: AY489187). The gene whose full cDNA length is 1 044 bp containing 3 exons and 2 introns is located in human chromosome 15q26.2, the cDNA encodes a novel protein of 105 amino acides with a theoretical molecular weight of 11.7 kD and isoelectric point of 10.09 which shares no significant homology with any known proteins in database. Real time PCR analysis of testis of different developmental periods revealed that SRG8 gene is significantly expressed in adult testis. The green fluorescent protein produced by pEGFP-C3/SRG8 was detected in the nucleus of HeLa cells after 24 h post-transfection. Cell cycle analysis showed that SRG8 can accelerate HeLa cells to traverse the S-phase and enter the G2-phase compared with the control without transfection of SRG8, which suggested that this gene plays an important role in the development of testis. The discovery of SRG8 showed that DDD combined with experiments is a feasible, time-saving strategy to identify new candidate genes for testis-specific development

Objective: To identify the resource suspected sperm of donor in human sperm bank and apply the parentage testing between the donor and his offspring., Methods: We took the 6 semen specimen of the donor involved and correspondently suspected semen as well as the semen of one volunteer and peripheral blood of his offspring. All specimens were amplified by PCR, and DNA fingerprint was detected by PAGE electrophoresis., Results: By DNA fingerprinting we discovered that the 5 suspected sperm samples came from corresponding donors and the other sample from another. The volunteer and his offspring were identified as consanguinity., Conclusion: We can identify the difference between sperm of donors and the suspected sperm of donors, and sperm of donors and the peripheral blood of their offspring exactly by DNA fingerprinting.

To understand molecular mechanism of spermatogenesis, two ESTs BG720564 and AI700454, were found from SPAG4 (sperm antigen 4), a gene related to dense fiber protein of outer membrane of the human sperm and mouse spermatocytes gene AK006225. The gap was filled by polymerase chain reaction, and a 1252 bp fragment was obtained. This 1252 bp fragment was named TSARG4 (testis and spermatogenesis related gene 4 (GenBank accession number AF401350). Its opening reading frame was 94-1233 bp, as was proved by RT-PCR in human testis. TSARG4 gene was located in 20q11.2, and the putative protein was 379 amino acid with a theoretical molecular weight of 43 081 and isoelectric point of 8.61. The homologies of amino acid sequences were 74% between TSARG4 and AK006225 gene and 45% between TSARG4 and SPAG4 gene, respectively. The human TSARG4 mRNA is expressed in a wide range of adult tissues, including testis, whereas the homologous mouse spermatocytes gene AK006225 is expressed specifically in the testis.