Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model

Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.     

In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars

The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25-ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocyte-complexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozen-thawed epididymal semen samples showed motility rates (72.2 +/- 5.6%) similar to those of spermatozoa in fresh semen (76.4 +/- 4.5%), while sperm motility decreased in frozen-thawed ejaculated semen (40.2 +/- 9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-cell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozen-thawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen.     

Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.     

Total reactive antioxidant potential and DNA fragmentation index as fertility sperm parameters

There is a growing evidence that oxidative stress play a major role in the etiology of defective sperm function including impaired morphology, motility, metabolism and fertility. The aim of the present study was to examine: 1/ total reactive antioxidant potential (TRAP) in seminal plasma; 2/ sperm DNA fragmentation index (DFI), 3/ sperm morphology and motility and 4/ cellular membrane integrity (hypoosmotic swelling test: HOS test) in patients attending in vitro fertilization/intracytoplasmatic sperm injection ( IVF/ICSI) program. According to the DFI value, the men were divided into: group 1 with DFI ≤15% (n=38) and group 2 with DFI ≥15% (n=37). Significant differences between the two groups were found in TRAP, sperm motility, morphology and concentration as well as HOS test scores. In group 1, DFI was negatively correlated with sperm motility and HOS test scores (p<0.05). The sperm morphology was positively correlated with sperm motility and HOS test scores in both groups. There was no correlation between TRAP and sperm chromatin fragmentation. Our results suggest that seminal plasma TRAP level may be a DFI independent parameter of sperm fertility.     

Increase of ICSI efficiency with hyaluronic acid binding sperm for low aneuploidy frequency in pig

The present study was designed to evaluate the ability of hyaluronic acid binding sperm (HABS) in increasing the efficiency of intracytoplasmic sperm injection (ICSI) in terms of the production of chromosomally normal porcine embryos. Porcine embryos were produced by in vitro fertilization (IVF), ICSI and ICSI using hyaluronic acid binding sperm (ICSI-HABS). Chromosome aneuploidy in sperm and embryos was evaluated using chromosome 1 submetacentric probe for fluorescence in situ hybridization (FISH) analysis. No significant differences were observed in the blastocysts rates (18.6, 23.6 and 23.8%) and cell numbers (61.8+/-12.5, 55.5+/-7.3 and 59.3+/-9.6) among embryos derived from IVF, ICSI, and ICSI-HABS. However, the frequency of normal diploidy in ICSI-HABS (75.5%) was significantly higher (P<0.05) than that in IVF (57.0%) and ICSI (68.2%). Embryos from ICSI-HABS showed significantly lower chromosome abnormality rate (P<0.05). Both ICSI and IVF embryos showed higher rates of polyploidy, and hence chromosomally abnormal embryos, in comparison to ICSI-HABS embryos. In addition, we investigated the chromosomal complement of porcine spermatozoa by FISH. The rate of chromosome number abnormality in porcine sperm was found to be 6.25% (70/1120). Thus, we conclude that the use of hyaluronic acid binding sperm is superior to morphological sperm selection for ICSI in producing chromosomally normal embryos and increasing the ICSI efficiency by lowering the aneuploidy frequency. Our results indicate that the selection of normal sperm with hyaluronic acid binding assay might help to reduce the early embryonic mortality due to chromosomal aneuploidy thereby increasing the success rate of embryo transfer technology in pigs.     

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21-39 for SMI and 50-61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2-10 and 23-40%, respectively). The incidences of sperm penetration were high (61-89% of inseminated oocytes) when the sperm were preincubated for 0-60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10-72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.     

Numération, mobilité, térastospermie et résultats en AMP

Since the advent of different methods of medically assisted procreation, investigations have increased in search of correlations between sperm parameters and fertilization potential of spermatozoa. This review reports the results of correlations between the three most commonly studied sperm characteristics (sperm count, motility, morphology) and the outcome of different methods procreation (AI, IVF, ICSI). Although sperm analysis remains the basic investigation for the evaluation of fertility, none of the three variables, taken individually, can predict the fertilizing potential of a sample because of the multifactorial character of fertilization. The contradictory results encountered sometimes in the litterature are partly due to non-standardized evaluation methods of sperm quality. Hence the necessity to standardize investigation techniques so as to use a common language and be able to compare results of different studies.     

Evaluation of chromosomal risk following intracytoplasmic sperm injection in the mouse

To investigate whether cytogenetic risks occur using the mouse intracytoplasmic sperm injection (ICSI) technique, the incidence of chromosome aberrations was compared in one-cell embryos produced by ICSI technique and those by conventional in vitro fertilization (IVF) technique. Spermatozoa were incubated in TYH medium for 1.5-2 h before IVF insemination. For the ICSI technique, spermatozoa were incubated in five different media: TYH, Hepes-buffered TYH (H-TYH), modified CZB (mCZB), Hepes-buffered mCZB (H-mCZB), and PB1 for 0.5 h, 2-2.5 h, and 6 h before injection into metaphase II oocytes. The incidence of IVF embryos with structural chromosome aberrations was 2%, whereas the occurrence of structural chromosome aberrations in ICSI embryos was dependent on the kind of medium and sperm incubation time. When spermatozoa were incubated in TYH medium for 2 h or more, the aberration rates in the resultant ICSI embryos (4%) were not significantly different from that of IVF embryos. However, there was a significant increase in aberration rates in ICSI embryos derived from spermatozoa that were incubated in other culture conditions (6%-28%). In addition, a time-dependent increase in aberration rates was found in ICSI embryos when H-TYH, H-mCZB, and PB1 were used for sperm incubation. There was no significant difference in incidence of aneuploidy between IVF and ICSI embryos. The chromosome analysis results of one-cell embryos were reflected by the performance of postimplantation embryo development. The causal mechanism of chromosome damage in ICSI embryos was discussed in relation to the plasma membrane cholesterol, the acrosome, and in vitro aging of spermatozoa.     

Low temperature storage of rhesus monkey spermatozoa and fertility evaluation by intracytoplasmic injection

The objective was to develop a sperm freezing procedure suitable for use in the propagation of valuable founder animals by assisted reproductive technologies. Here, we report a comparison of processing methods by measuring the motility of fresh and frozen-thawed rhesus monkey spermatozoa and fertility via intracytoplasmic spermatozoa injection (ICSI) of sibling oocytes. Washed spermatozoa were frozen in straws or in pellets using different cryoprotective media and processed post-thaw with or without a density gradient centrifugation step. Among the four study series, motility post-thaw was improved with density gradient centrifugation (17-24% versus 75%, P<0.01) achieving levels similar to fresh spermatozoa. Spermatozoa injected oocytes (total n=377) were co-cultured on BRL cells and observed for fertilization and development. With spermatozoa frozen in straws in liquid nitrogen vapors, the fertilization rate after ICSI was lower than with fresh spermatozoa (40-44% versus 77-86%, P<0.05), even with the Percoll-enriched fraction that exhibited robust motility. In contrast, somewhat slower freezing of spermatozoa in pellets on dry ice supported fertilization rates (73%) that were similar to the fresh counterpart. Developmental rates of fertilized eggs were similar in all experiments. A total of 106 embryo transfers has resulted in the first primate born after ICSI with F/T ejaculated spermatozoa plus 22 other infants to date. Additionally, a 3-4 h incubation after thawing improved the fertilization rate with spermatozoa from a male with poor post-thaw recovery of sperm motility. In conclusion, an acceptable fertilization rate after ICSI with motile, frozen-thawed primate spermatozoa was observed comparable to that obtained with fresh spermatozoa allowing small quantities of competent spermatozoa to be used with ICSI to facilitate propagation of desirable primate genotypes.     

Normospermic versus teratospermic domestic cat sperm chromatin integrity evaluated by flow cytometry and intracytoplasmic sperm injection

Teratospermia (>60% of morphologically abnormal spermatozoa) is well documented in felids. Even morphologically normal spermatozoa from teratospermic ejaculates have reduced ability to undergo tyrosine phosphorylation, acrosome react, and bind and penetrate oocytes compared with normospermic (<40% abnormal spermatozoa) counterparts. However, it is unknown whether fertilization deficiencies originate at a nuclear level. This study examined whether fertilization failure also was attributable to abnormal sperm chromatin, using the sperm chromatin structure assay (SCSA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Aliquots of unprocessed and swim-up-processed (to isolate morphologically normal spermatozoa) spermatozoa from teratospermic and normospermic domestic cats were analyzed by the flow cytometric SCSA. Swim-up-processed sperm were incubated with in vivo-matured oocytes or used for ICSI. Teratospermic ejaculates expressed more (P < 0.05) chromatin heterogeneity (abnormal chromatin structure) than their normospermic counterparts, both in unprocessed and swim-up-processed samples. Fertilization success in vitro was higher (P < 0.05) from normo- compared with teratospermic inseminates. Similar (P > 0.05) proportions of oocytes fertilized after ICSI using spermatozoa from normo- and teratospermic cats. Results reveal that teratospermia in the cat is expressed at the nuclear level as increased sperm chromatin heterogeneity, but ICSI showed that this does not apparently affect fertilization rates if the zona pellucida and oolemma can be bypassed.     

Influence of sperm:oocyte ratio during in vitro fertilization of in vitro matured cumulus-intact pig oocytes on fertilization parameters and embryo development

The present study was conducted to evaluate the influence of sperm:oocyte ratio during in vitro fertilization (IVF) of in vitro matured cumulus-intact oocytes on fertilization parameters and embryo development in pigs. In vitro matured oocytes surrounded by intact cumulus cells (COC) were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000:1, 3000:1, 4000:1, 6000:1, and 8000:1). Denuded oocytes inseminated with 2000 frozen-thawed spermatozoa:oocyte were the control group. A total of 2546 oocytes in five replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture (EC) medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The penetration rate increased in the COC groups with the sperm:oocyte ratio, reaching the highest rates with 8000:1 spermatozoa:oocyte (72.1 +/- 6.5%), similar to the control (73.5 +/- 3.5%). However, the monospermy was highest with the lower spermatozoa:oocyte rates (82.6-94.8%) and decreased drastically (P<0.05) in the COC group fertilized with 8000 sperm:oocyte (36%). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) showed no difference among the COC groups (20-30%) but they were significantly lower (P<0.007) than those obtained by the control group (43.7 +/- 2%). Embryo development was highest in the control group (58% for cleavage and 23% for BF) but not significantly different with the 6000 and 8000 sperm:oocyte COC groups (47 and 50% for cleavage and 19 and 17% for BF, respectively). These results indicate that the use of COC for IVF involves a drop in the efficiency of the fertilization and the necessity to increase the frozen-thawed sperm:oocyte ratio three to four times more to obtain similar embryo development to denuded oocytes.     

L’exploration du sperme pathologique et de son pouvoir fécondant a-t-elle encore un intérêt à l’heure de l’ICSI?

The semen analysis in the assisted reproductive technology (ART) is mainly subjective, and requires an extensive training. However, other sophisticated techniques can be used in order to evalute functionnal state of sperm cell. Today, in vitro fertilization (FIV) as well as the intra-cytoplasmic sperm injection (ICSI) are considered as the adequate mean by which the sperm quality can be evaluated. Should all men undergo all tests? One must think not only in terms of efficiency but also of cost. No doubts that a full work up, in all men will detect the high risk husbands in IVF. But to achieve this goal a lot of work is required. Most IVF AND ICSI programs use a simple wash, swim-up or Percoll gradients to prepare sperm for insemination. The goal of these techniques is to remove debris, cells bacteria and to allow the selection of morphologically normal motile spermatozoa. These normal motile spermatozoa are mainly selected according to their active motility (swim up) or specific gravity (percoll gradient) which reflects the degree of head condensation.     

Apoptose dans le sperme éjaculé: Revue

It has become clear in recent years that programmed cell death occurs spontaneously in the cycle of the seminiferous epithelium. Although apoptosis is a key phenomenon in the control of sperm production, the existence and role of apoptosis in ejaculated sperm cells remain controversial. Apoptosis — as determined by DNA fragmentation and ultrastructural analysis — is abnormally frequent in the sperm cells of the ejaculate of sterile men. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa and the consequences of this DNA damage when apoptotic spermatozoa are used for ICSI. Percentages of DNA fragmentation in human ejaculated sperm are correlated with fertilization rates after IVF or ICSI assay. Detection of DNA fragmentation in human sperm could provide additional information about the biochemical integrity of sperm and may be used in future studies for fertilization failures not explained by conventional sperm parameters. However, the analysis of new markers of apoptosis (Fas, ANNEXINE V) and molecular mechanisms is now necessary to assess the role of apoptosis in human ejaculated sperm cells.     

Relationships between bacteriospermia,DNA integrity,nuclear protamine alteration,sperm quality and ICSI outcome

The Aim of this study was to evaluate the effects of bacteriospermia on human sperm parameters, nuclear protamines, DNA integrity and ICSI outcome in patients enrolled for ICSI treatment. 84 unselected couples consulting in infertility and obstetrics clinic and enrolled for ICSI treatment were included in this study. The semen specimens were screened bacteriologically; semen and sperm parameters were also evaluated according to WHO guidelines. DNA integrity, protamines concentration and protamine deficiency were estimated by TUNEL assay, AU-PAGE and Chromomycin (CMA3) respectively. The results of this study revealed that 34.52% of studied semen samples were infected with bacteria. The isolated bacteria were identified as Staphylococcus aureus, Staph. epidermidis, Staph. haemolyticus, Escherichia coli, Enterococcus faecalis and Streptococcus agalactiae. Bacteriospermia had a significant (p?<?.010) negative effect on sperm parameters; concentration, motility, progressive motility and chromatin condensation. Moreover, high DNA fragmentation with low P1 and P2 concentrations were noticed in infected patients in comparison to non-infected patients but non-significant. Also, the fertilization rate decreased significantly (p?<?.05) with infected patients. In conclusion: bacteriospermia has significant negative effect on sperm quality and fertilization rate in patients who underwent ICSI treatment.     

Bulky DNA adducts in human sperm: relationship with fertility,semen quality,smoking, and environmental factors

The integrity of DNA of spermatogenic cells can be affected by endogenous and exogenous genotoxic factors. Resulting DNA damage in spermatozoa may significantly contribute to impaired fertility. Here, the 32P-postlabeling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 males, either healthy donors or patients with an impaired fertility. When all donors were analyzed, the levels of bulky DNA adducts was 1.2-fold higher in smokers than in non-smokers, but the difference was not statistically significant (P=0.054). However, a statistically significant difference existed between current smokers and never smokers among the healthy individuals (1.7-fold increase, P=0.008). No correlation between alcohol or coffee consumption and sperm DNA adducts was found. The levels of DNA adducts in sperm seemed to be unaffected by environmental and occupational factors. On the other hand, groups of healthy persons and patients with male-factor infertility differed significantly with respect to the level of bulky DNA adducts (P=0.012). A significant negative correlation between DNA adducts and sperm concentration or sperm motility existed among patients with an impaired fertility (n=93; P<0.029, r(S)=-0.225). These results suggest that DNA adducts in sperm cells can be applied as potential biomarkers in studies of human infertility.     

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.     

Osteopontin reduces polyspermy during in vitro fertilization of porcine oocytes

This study was designed to determine the role of osteopontin (SPP1) in in vitro fertilization (IVF) in swine. The initial objective was to evaluate the effect of various concentrations of SPP1 (0, 0.001, 0.01, 0.1 and 1 microg/ml) on spermatozoa and oocytes during IVF. The results demonstrate that SPP1 reduced the rate of polyspermy in a dose-dependent manner (P < 0.05). SPP1 also reduced both the number of sperm in oocytes as compared to the control and the number of spermatozoa bound to the zona pellucida (ZP) (P < 0.05). High doses of SPP1 (1 microg/ml) reduced penetration and male pronucleus formation as compared to the control (P < 0.05). Interestingly, compared to the control group, medium doses of SPP1 increased fertilization efficiency (42.6% and 44.6% vs. 31.6%; P < 0.05), representing a 41% improvement for 0.1 microg/ml SPP1). The ZP of 0.1 microg/ml SPP1-treated oocytes was more difficult to digest than control oocytes (P < 0.05). The percentage of acrosome-reacted spermatozoa bound to the ZP during IVF increased after 4 h of 1.0 microg/ml SPP1 treatment compared to 0 or 0.1 microg/ml SPP1. SPP1 did not have an effect on sperm motility, progressive motility, and sperm viability. To confirm that the reduction of polyspermy was specific to SPP1, a mixture of pregnancy-associated glycoproteins was included in the IVF protocol and shown to have no effect on polyspermy. Furthermore, Western blotting demonstrated that a 50-kDa SPP1 form was present in the oviducts on Days 0, 3, and 5 in pregnant and nonpregnant gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5. The current study represents the first report to demonstrate that SPP1 plays an important role in the regulation of pig polyspermic fertilization; it decreases polyspermy and increases fertilization efficiency during IVF.     

The treatment of obstructive azoospermia by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) allows the treatment of virtually every type of male infertility. Unlike in vitro fertilization (IVF), its success does not depend on sperm concentration, motility or morphology and most of the physical barriers to fertilisation are by-passes. Since ICSI does not require strongly motile sperm, its use has now been expanded to incorporate immature sperm from the testes and epididymides. Successful fertilisation, pregnancies and healthy babies have all been reported. However, concerns about the safety of ICSI remain due to its short clinical history and the lack of testing on animal models. Male fertility potential for assisted reproduction by ICSI cannot be measured by conventional parameters. Sperm DNA integrity is increasingly recognised as a more useful indicator. Studies have shown that sperm with higher levels of DNA damage have lower fertilisation rates after IVF and ICSI. Sperm with DNA damage above a certain threshold are associated with a longer time to conceive in otherwise apparently fertile couples and a higher miscarriage rate. DNA damage has been shown to be associated with impaired embryo cleavage. Our group has shown that sperm DNA from testicular sperm is less fragmented than that from epididymal sperm and suggest its preferred use in ICSI. In addition to nuclear (n) DNA we also assessed the quality of mitochondrial (mt) DNA from testicular sperm from men with obstructive azoospermia undergoing ICSI. We observed that couples achieving a pregnancy had both less mtDNA deletions and less nDNA fragmentation. We found inverse relationships between pregnancy and sperm mtDNA deletion numbers, size and nDNA fragmentation. No relationships were observed with fertilisation rates. With this knowledge, we designed an algorithm for the prediction of pregnancy based on the quality of sperm nDNA and mtDNA. Each year 40,000 men have a vasectomy in the UK but every year 2500 request a reversal to begin a second family. For such men, vasectomy reversal has recently been replaced in part by testicular biopsy via fine-needle testicular sperm aspiration (TESA) or percutaneous epididymal sperm aspiration (PESA) performed at an outpatient clinic and subsequently used in ICSI. Since these were previously fertile men it has been assumed that they had ‘fertile’ sperm. However the assited conception success rates of these mens partners has not been assessed until recently. We have shown a significant reduction in the clinical pregnancy rates in the partners of men who had had a vasectomy ≥10yrs previously. There is also evidence to suggest that spermatogenesis is significantly impaired in vasectomised men. Marked decreases in spermatocytes, spermatids and spermatozoa have been observed. We have found this to be associated with concomitant increases in apoptotic markers, such as Fas, FasL and Bax. The quality of the remaining sperm is also compromised. Sperm DNA from vasectomized men shows substantial damage which increases with time after surgery. This new use of ICSI will be discussed.     

Human sperm acrosomal status,acrosomal responsiveness,and acrosin are predictive of the outcomes of in vitro fertilization: A prospective cohort study

The sperm acrosome reaction (AR) is a physiological secretory course of membrane fusion and hydrolytic enzymes, as well as matrix protein release, enabling spermatozoa to penetrate the egg surroundings. An instable acrosomal status before a specific stimulus, insufficient acrosomal responsiveness, or inadequate enzymatic activity of acrosomal content can be detrimental to male fertility. This prospective cohort study was designed to determine whether three human sperm acrosome evaluation parameters—including spontaneous AR rate, AR after calcium ionophore A23187 challenge (ARIC) rate, and modified Kennedy acrosin activity—can predict fertilization outcomes in vitro and are correlated with male characteristics. A total of 485 eligible couples undergoing in vitro fertilization (IVF) therapy were included in two phases of this study. In a ‘construction phase’, three acrosome evaluation parameters were determined simultaneously in 132 cases, whereas in a ‘validation phase’, the spontaneous AR rate was determined in 353 cases. The results of the ‘construction phase’ revealed that the spontaneous AR rate was the only significant predictor of fertilization outcome (unadjusted odds ratio [OR]?=?0.68, 95% confidence interval [CI]: 0.53–0.88, P?=? 0.003; adjusted OR = 0.64, 95% CI: 0.43–0.95, P?=? 0.03), and the cut-off value for total fertilization failure (TFF) prediction, determined by ROC curve analysis, was 9.91%; higher acrosin activity was shown to predict a higher fertilization rate only when patients were divided into groups (≥25 μIU/10⁶ spermatozoa, 14–25 μIU/10⁶ spermatozoa, <14 μIU/10⁶ spermatozoa). The spontaneous AR rate was negatively correlated with sperm motility, forward progression motility, and normal morphology; modified Kennedy acrosin activity was positively correlated with normal morphology; and the ARIC rate was not correlated with any of the male characteristics. A similar result was obtained for the spontaneous AR rate in the ‘validation phase’, and the cut-off value in predicting TFF was calibrated for 9.52%. Clinically, patients can voluntarily choose spontaneous AR rate alone or in combination with modified Kennedy acrosin activity to predict TFF, and early rescue intracytoplasmic sperm injection (ICSI), half ICSI, or full ICSI should be considered in advance for men with spontaneous AR rates ≥9.52% or spontaneous AR rates ≥9.52% and AE activities <25 μIU/10⁶ spermatozoa.     

Characterization of semen collected from beagles and captive Japanese black bears (Ursus thibetanus japonicus)

This study characterized semen collected from the Japanese black bear, Ursus thibetanus aponicus, to provide information on semen cryopreservation for artificial breeding. Preliminary studies using a beagle dog as the model species showed that sperm concentration and total sperm count were lower in semen collected by electroejaculation than in semen collected by digital manipulation, but that sperm motility, viability and morphology were similar. Characterization of semen obtained from Japanese black bears by electroejaculation under general anesthesia revealed that semen volume and total number of spermatozoa collected were lower; but that sperm concentration, motility, viability and morphology were equivalent to those reported in other ursids. When semen was collected via a catheter inserted into the urethra during the stimulation for ejaculation, the sperm concentration, total sperm count and motility were relatively higher than when semen was collected directly in a test tube. Specific normal semen characteristics (mean +/- SEM) were pH, 7.6 +/- 0.0; volume, 0.212 +/- 0.038 mL; sperm concentration, 361 +/- 100 x 10(6)/mL; total sperm count, 84.0 +/- 32.2 x 106; +++ motility, 30 +/- 5%; motility, 77 +/- 3%; viability 77 +/- 2%; and abnormal morphology, 11+/- 2%. These results suggest that semen can be collected from Japanese black bears by electroejaculation.