OBJECTIVETo study the effect of direct fumigation on the post-thaw recovery rate of cryopreserved spermatozoa, and to search for a best method for human sperm cryopreservation.

METHODSWe collected semen samples from 100 donors conforming to the normal reference values in WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed), divided them into two groups, and subjected them to cryopreservation by programmable freezing (Group A) and direct fumigation (Group B), respectively. We detected the progressive motility of pre-freezing and post-thaw sperm with a computer-assisted semen analyzer, and compared the effects of the two methods on the functional integrity of sperm membrane and the rate of abnormal sperm using the percentage of hypo-osmotic swelling sperm and modified Papanicolaou staining.

RESULTSStatistically significant differences were found in post-thaw sperm progressive motility between the Groups A and B ([34.0 +/- 18.4]% vs [43.0 +/- 19.5]%, P<0.05), both remarkably decreased as compared with pre-freezing ([57.0 +/- 16.7]%, P<0.05). Such differences were also found in the post-thaw recovery rate of progressively motile sperm between the two groups ([52.2 +/- 20.6]% vs [67.1 +/- 20.0]%, P<0.05). The post-thaw percentage of hypo-osmotic swelling sperm was obviously decreased in both Groups A and B ([67.1 +/- 11.1]% and [70.6 +/- 10.0]%) in comparison with pre-freezing ([84.5 +/- 7.5]%, P<0.05), with significant differences between A and B (P<0.05). However, the rate of sperm abnormality was evidently increased in Groups A and B ([85.0 +/- 8.7% and [85.7 +/- 9.1]%), significantly higher than pre-freezing ([77.8 +/- 9.6]%, P<0.05), but with no significant differences between A and B (P>0.05).

CONCLUSIONDirect fumigation is superior to programmable freezing for its easier operation, wider application, and higher sperm recovery rate.

Adult ; Cryopreservation ; methods ; Humans ; Male ; Semen Analysis ; Semen Preservation ; methods ; Sperm Motility ; Spermatozoa ; Young Adult

OBJECTIVETo investigate the influence of different methods of semen preservation and processing on sperm DNA integrity.

METHODSWe collected semen samples from 100 normozoospermic male volunteers and, following homogeneous mixing, preserved them by means of snap freezing, slow freezing, or at the room temperature for 4 and 24 hours. Meanwhile we processed the semen by washing, swim-up, and density gradient centrifugation (DGC). Then we obtained the sperm DNA fragmentation index (DFI) by sperm chromatin dispersion test and measured total sperm motility and DFI after cultured for 24 hours following processing.

RESULTSThe sperm DFIs after 4 hours of preservation by snap freezing, slow freezing, and at the room temperature were (27.3 ± 6.4)%, (26.9 ± 6.1)%, and (24.7 ± 6.8)%, respectively, and that after preserved at the room temperature for 24 hours was (35.6 ± 9.0)%, with statistically significant differences between the first three and the 24-hour room temperature preservation groups (P < 0.05) but not among the former three groups (P > 0.05). The sperm DFI was significantly higher in the samples processed by washing ([13.7 ± 2.0]%) than in those processed by swim-up ([9.1 ± 1.3]%) and DGC ([8.0 ± 2.5]%) (P < 0.05), and it was the lowest in the DGC group after 24-hour culture ([11.5 ± 4.2]%) as compared with the other groups (P < 0.05).

CONCLUSIONSperm DNA integrity is influenced by different semen preservation conditions and processing methods.

Centrifugation, Density Gradient ; DNA Fragmentation ; Humans ; Male ; Semen ; Semen Analysis ; Semen Preservation ; methods ; Sperm Motility ; Spermatozoa ; cytology

OBJECTIVETo search for a method for the precise measurement of human semen volume so as to provide reliable evidence for clinical semen analysis.

METHODSThe volumes of 492 semen samples collected from 137 donors by Zhejiang Human Sperm Bank were measured respectively by three different methods including electronic balance weighing, volumetric cylinder measuring, and combination of the two methods above. With the combined measuring method, the semen weight was first obtained by electronic balance weighing, then the semen density determined by volumetric cylinder measuring, and lastly the semen volume figured out by a formula. Paired sample t-test was used to compared the combined method with electronic balance weighing and volumetric cylinder measuring.

RESULTSThe mean volume of the 492 semen samples obtained by the combined measuring method was (3.46 +/- 1.17) ml, significantly lower than (3.75 +/- 1.21) ml from electronic balance weighing (P < 0.05) and markedly higher than (3.22 +/- 1.16) ml from volumetric cylinder measuring (P < 0.05). The mean semen density of the 492 samples was (1.0928 +/- 0.0761) g/ml, and the mean weight of the residual semen in the container used in volumetric cylinder measuring was (0.269 +/- 0.122) g.

CONCLUSIONSemen volume measured by electronic balance weighing is higher while that obtained from volumetric cylinder measuring is lower than the actual value. An accurate semen volume can be achieved by the combined measuring method, which, therefore, deserves to be widely used both clinically and in researches.

Adult ; Body Weights and Measures ; Humans ; Male ; Semen ; Semen Analysis ; methods ; Young Adult

Mycoplasma species (spp.) are bacteria that are difficult to detect. Currently, the polymerase chain reaction (PCR) is considered the most effective diagnostic tool to detect these microorganisms in both human and veterinary medicine. There are 13 known species of human Mycoplasma and 15 species of canine Mycoplasma. Owing to the difficulties in identifying the individual species of Mycoplasma, there is a lack of information regarding which species are saprophytic and which are pathogenic. The prevalence of the individual species is also unknown. In addition, in both humans and dogs, the results of some studies on the impact of Mycoplasma are conflicting. The presence of Mycoplasma spp. on the epithelium of reproductive tract is often associated with infertility, although they are also detected in healthy individuals. The occurrence of Mycoplasma spp. is more common in dogs (even 89%) than in humans (1.3%-4%). This is probably because the pH of a dog's genital is more conducive to the growth of Mycoplasma spp. than that of humans. Phylogenetically, human and canine Mycoplasma are related, and majority of them belong to the same taxonomic group. Furthermore, 40% of canine Mycoplasma spp. are placed in common clusters with those of human. This suggests that species from the same cluster can play a similar role in the canine and human reproductive tracts. This review summarizes the current state of knowledge about the impact of Mycoplasma on canine and human male fertility as well as the prospects of further development in this field.
Humans ; Dogs ; Male ; Animals ; Mycoplasma/genetics* ; Infertility ; Semen Analysis ; Polymerase Chain Reaction/methods* ; Prevalence ; Semen/chemistry*

The analysis of sperm morphology can be used to evaluate sperm fertilizing ability and spontaneous conception status, and especially the overall analysis of the sperm head, neck and tail, along with the patient's living habits, occupation and clinical manifestations, may contribute to the primary diagnosis of the patients potentia generandi. It can also be employed to assess the effects of the treatment of semen samples. Although oocyte fertilization can be achieved by the technologies of intracytoplasmic sperm injection (ICSI), motile sperm organelle morphology examination (MSOME) and intracytoplasmic morphologically selected sperm injection (IMSI) regardless of sperm morphology and / or motility, which may somewhat weaken the clinical application of sperm morphology analysis, the standardized procedure and the practice of quality control for the analysis of sperm morphology can significantly improve the accuracy of its results and largely promote its clinical application. Therefore, it is of positive necessity as well as clinical application value to perform sperm morphology analysis in andrology laboratories, reproductive centers, sperm banks and the department of laboratory medicine.
Humans ; Male ; Semen Analysis ; Sperm Injections, Intracytoplasmic ; methods ; Sperm Motility

OBJECTIVETo investigate the effect of pre-freezing equilibration on the cryo-survival of human sperm and to optimize the protocol of direct fumigation for the freeze-thawing of human sperm.

METHODSWe collected 50 semen samples from healthy donors, each subjected to cryopreservation with 3 different methods: non-equilibration freezing (Group A), 10-min equilibration at room temperature before freezing (Group B), and 10-min equilibration at 4 degrees C before freezing (Group C). We examined all the post-thaw semen samples by computer-assisted semen analysis for the sperm motility parameters, and detected the sperm vitality and deformity index (SDI).

RESULTSThe recovery rate of progressive sperm motility was (61.88 +/- 16.94)% in Group C, remarkably higher than in A ([48.61 +/- 16.44]%) and B ([49.41 +/- 13.77]%) (P < 0.05), but with no significant difference between the latter two. And there were no significant differences in sperm vitality and SDI among the three groups.

CONCLUSIONTen-minute equilibration at 4 degrees C before freezing can evidently improve the progressive motility of sperm in addition to its advantages of easy operation and controllable experimental condition.

Adult ; Cryopreservation ; methods ; Humans ; Male ; Semen Analysis ; Semen Preservation ; methods ; Sperm Banks ; Sperm Count ; Sperm Motility ; Young Adult

Fertility preservation is a hotspot of research in reproductive medicine, and that of male adolescent cancer patients is drawing even more attention from reproductive and oncologic clinicians. Both cancer and its treatment can decrease semen quality and even induce irreversible damage to fertility. Sperm cryopreservation is an effective method for fertility preservation. In the past few years, marked advances have been made in the cryopreservation, transplantation, and in vitro culture of testis tissue and stem spermatogonial cells. Although still experimental, these approaches may offer some options to those with no mature sperm in the testis. Unfortunately, very few people know and participate in the studies of fertility preservation and the utilization rate of cryopreserved sperm remains low. Therefor reproductive physicians and oncologists are required to make more efforts to search for effective fertility preservation methods for male adolescent cancer patients.
Adolescent ; Cryopreservation ; Fertility Preservation ; methods ; Humans ; Male ; Neoplasms ; therapy ; Semen Analysis ; Semen Preservation ; methods ; Spermatogonia ; Testis ; cytology

OBJECTIVETo investigate the semen quality of cancer patients and search for a better way of sperm cryopreservation for them.

METHODSWe retrospectively analyzed the quality of the semen from 43 cancer patients under cryopreservation in the Sperm Bank of Zhejiang Province, and compared the semen parameters between the cancer patients and 248 normal donors as well as between the testicular cancer cases (n=22) and non-testicular cancer cases (n=21).

RESULTSThe cancer patients exhibited significantly lower semen quality than the normal donors as in sperm concentration (60.90 x 10(6)/ml vs 74.27 x 10(6)/ml), progressive motility (41.07% vs 51.79%), and recovery rate (49.98% vs 57.33%) (all P <0.05). Furthermore, the progressive sperm motility and sperm recovery rate after freezing were significantly decreased in the testicular cancer cases (15.68% and 42.81%) than in the non-testicular cancer cases (28.36% and 57.53%) (both P <0.05).

CONCLUSIONSemen quality declines in cancer patients, and therefore early sperm cryopreservation is essential for them. Due to the poor sperm motility and recovery rate of testicular cancer patients after freezing, further investigation is required on the improvement of sperm cryopreservation methods.

Adult ; Cryopreservation ; methods ; Humans ; Male ; Neoplasms, Germ Cell and Embryonal ; Semen ; Semen Analysis ; Semen Preservation ; methods ; Sperm Count ; Sperm Motility ; Testicular Neoplasms

Semen stain identification is one of the crucial tasks for collection of criminal evidence by forensic techniques. Substances such as DNA and RNA contained in semen stains can serve as a source of personalized evidence targeting the suspect. Therefore, semen stain identification is vital to inferring the case attributes and the facts of the crime. The conventional methods of forensic stain identification focus on the detection of specific-function protein and/or high-content protein, such as alkaline phosphatase and PSA. Although the specificity of such protein markers is relatively high, these methods yield a limited rate of success for several factors, including poor stability, low sensitivity of the target protein, and possible subjectivity of the performer. In order to overcome these limitations, new technologies such as Raman spectroscopy, mass spectrometry for protein markers, sperm-specific aptamer, mRNA, microRNA, and DNA methylation assays have been studied and recommended by many investigators. These new technologies are paving a new ground for personalized trace analysis and even for detection of over-timed specimens.
DNA ; analysis ; DNA Methylation ; Forensic Medicine ; methods ; Humans ; Male ; MicroRNAs ; analysis ; RNA, Messenger ; analysis ; Semen Analysis ; methods ; Sensitivity and Specificity ; Spermatozoa

OBJECTIVE: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. METHODS: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. RESULTS: The sperm DFI showed a significant correlation (r=–0.347, p<0.001) with sperm motility and morphology (r=–0.114, p<0.05) but not with other semen parameters. The DFI (11.5%±2.0%) of semen samples was significantly reduced by DGC (8.1%±4.1%) or MACS alone (7.4%±3.9%) (p<0.05). The DFI was significantly further reduced by a combination of DGC and MACS (4.1%±1.3%, p<0.05). Moreover, the combination of DGC and MACS (1.6%±1.1%, p<0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC (4.4%±3.2%) or MACS alone (3.4%±2.2%). CONCLUSION: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.
Apoptosis ; Centrifugation* ; Centrifugation, Density Gradient ; Chromatin* ; DNA Fragmentation ; DNA* ; Methods ; Product Packaging* ; Semen ; Semen Analysis ; Sperm Motility ; Spermatozoa*