Macrophage Scavenger Receptor(s) and Oxidatively Modified Proteins in Endometriosis

Objective: To determine whether cultured human peritoneal macrophages have functional scavenger receptor(s) and whether activation of macrophages in endometriosis may involve an increase in scavenger receptor activity.

Design: A controlled clinical study comparing peritoneal fluid (PF) macrophages of women with endometriosis and controls without endometriosis.

Setting: Women undergoing laparoscopic evaluation and treatment in a tertiary medical center.

Patient(s): Twenty-one women undergoing evaluation for pelvic pain or infertility and 10 women undergoing elective laparoscopic tubal ligation.

Intervention(s): None.

Main Outcome Measure(s): Evidence for functional macrophage scavenger receptor and evidence of ligands for the scavenger receptor in PF.

Result(s): Peritoneal macrophages of women with endometriosis degrade significantly more endothelial cell-low density lipoprotein (EC-LDL) and copper-oxidized LDL (Cu-LDL) than native LDL. Macrophages of women with endometriosis also incorporate more labeled oleic acid into cholesteryl ester in the presence of oxidized LDL (Ox-LDL) than in the presence of native LDL. Western blot analysis demonstrates the presence of adducts between lipid peroxidation products and proteins in PF of patients with and without endometriosis. The PF of women with endometriosis competes with labeled Ox-LDL for uptake by mouse peritoneal macrophages in a dose-dependent manner.

Conclusion(s): We demonstrate for the first time that human macrophages have functional scavenger receptor(s) and that activation of macrophages in endometriosis involves an increase in scavenger receptor activity. Two lines of evidence indicate the presence of ligands for the scavenger receptor in PF.     

Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity

Objective: To investigate a method of assisted activation of human oocytes for the treatment of infertility resulting from globozoospermia associated with deficient oocyte activation capacity.

Design: The mouse oocyte activation test was used to analyze the oocyte activation capacity of the sperm cells of a patient with globozoospermia. Fresh donor human oocytes were used for determining the most appropriate procedure for oocyte activation.

Setting: Infertility Center, University Hospital of Ghent.

Patient(s): A couple with infertility resulting from globozoospermia.

Intervention(s): Intracytoplasmic sperm injection, assisted oocyte activation, and embryo transfer.

Main Outcome Measure(s): Oocyte activation and fertilization rates, implantation, and pregnancy.

Result(s): Deficiency in oocyte activation capacity was found in the sperm of a patient with globozoospermia. This deficiency was proven by the mouse oocyte activation test and was confirmed further by lack of activation of human oocytes after simple sperm injection. Only human oocytes that were injected with sperm and subjected to calcium chloride and ionophore treatment underwent activation. Transfer of embryos obtained by this procedure of assisted oocyte activation resulted in an ongoing pregnancy.

Conclusion(s): Assisted oocyte activation of human oocytes is useful when globozoospermia is associated with absence of oocyte activation capacity in the sperm. These cases can be identified by the mouse oocyte activation test.     

Inhibitory effects of monoclonal sperm antibodies on the fertilization of mouse oocytes in vitro and in vivo

The inhibitory effects of monoclonal antibodies on the fertilization of mouse oocytes were evaluated in vitro and in vivo. Among the 40 sperm-specific monoclonal antibodies that had been examined, nine showed significant inhibition of the fertilization of mouse oocytes in vitro. MS 204 was shown to cause a high incidence of penetration of the zona pellucida of mouse eggs by multiple sperm, when the sperm concentration for insemination exceeds 1 X 10(5)/ml. This antibody prevented further penetration of the vitelline membrane by sperm. On the other hand, sperm penetration to zona was inhibited in the presence of MS 207. However, neither MS 204 nor MS 207 caused significant inhibition of penetration of zona-free mouse or hamster eggs by sperm. MS 204 and MS 207 were also found to inhibit the fertilization of mouse oocytes in vivo and embryo development in vitro, when superovulated mice were injected intraperitoneally with given doses of antibodies prior to the mating. Further in vitro culture of the recovered 2-cell oocytes revealed little or no further embryo development beyond two- to four-cell stages. In the controls, greater than 80% of the retrieved oocytes were fertilized and successively developed to the blastocyst stages in vitro when the ascites fluid from NS-1 cells was administered. Two of the monoclonal antibodies generated against human sperm antigens, HS 11 and HS 63, were shown to cross-react specifically with mouse sperm acrosomal antigens and also inhibited the fertilization of mouse oocytes in vitro and in vivo. The results of this study suggest that some monoclonal antibodies to sperm acrosomal antigens exhibit strongly inhibitory effects on the in vitro and in vivo fertilization of mouse oocytes as well as subsequent development of early embryos. As a comparative control, rabbit antisera against sperm-specific enzymes, lactate dehydrogenase-X and 3-phosphoglycerate kinase-2, showed little or no inhibition on the fertilization of mouse oocytes in vitro or in vivo or the subsequent embryo development.     

Generation of mouse oocyte monoclonal isoantibodies: their effects and those of antisperm monoclonal antibodies on in vitro fertilization

Monoclonal isoantibodies to mouse oocyte antigens were generated by modified hybridoma techniques similar to those described for mouse sperm monoclonals. Following isoimmunization with mouse oocytes and cell fusion, hybrid cells were cultured initially in a semi-solid medium containing methylcellulose. Seven to ten days after cell fusion about 350 hybrid clones were recovered for subculture. By an indirect immunofluorescence assay using frozen or fresh mouse oocytes, twenty hybridomas were shown to produce antibodies that bind to various oocyte components including antigens of the zona pellucida. However, they did not cross-react with mouse spermatozoa or lymphocytes.A system was established to evaluate whether monoclonal antibodies to gamete-specific antigens have any inhibitory effects on the fertilization of mouse oocytes in vitro. A monoclonal antibody against zona antigen(s), ME 56, was shown to block fertilization of mouse oocytes via the inhibition of sperm binding to the zona pellucida. On the other hand, three out of four antibodies reacting with mouse sperm acrosomes were also inhibitory to mouse in vitro fertilization, perhaps mainly due to the inhibition of sperm acrosomal reactions. Using a sodium dodecylsulfate gel/protein blot radioimmunobinding method, the molecular weight of zona antigen(s) that react with ME 56 was determined to be in the range of 95,000, whereas that of the acrosomal antigen(s) reacting with the fertilization-inhibiting antibody, MS 207, was about 30,000. The results of this preliminary study suggest that monoclonal antibodies to certain gamete antigens can be a valuable tool for the analysis of sperm-egg interactions during the fertilization processes.     

Assisted fertilization of mouse oocytes and preliminary results for human oocytes using zona drilling

The zona-drilling procedure was investigated in mouse oocytes prior to a study on human oocytes. The procedure involved the injection of 5-nl volumes of acidic Hepes-buffered medium at pH 2.5 using a microinjection instrument. Zona-drilled mouse oocytes had significantly higher rates of fertilization (60/99; 61%) than zona-intact oocytes (6/103; 6%) at an insemination concentration of 1×10⁴ sperm/ml (P<0.001). The procedure did not induce parthenogenetic activation of oocytes and more than 97% of zygotes developed to the blastocyst stage. A similar rate of live progeny was observed when zona-drilled (38.0%) and control embryos (38.5%) were transferred to pseudopregnant recipients. Chromosome analyses were performed on zona-intact, zona-free, and zona-drilled oocytes inseminated with varying concentrations of sperm and analysed at the first cleavage division. Zonafree oocytes had high rates of polyploidy (40%) with varying insemination numbers but the zona-drilled oocytes did not reveal significant increases in the rate of polyploidy or aneuploidy when compared to controls. In the human studies, zona-drilled oocytes achieved higher rates of fertilization than zona-intact oocytes, with sperm numbers as low as 1×10⁴/ml (6/8; 75%). Polyspermic fertilization was observed in 1/2 and 2/6 of fertilized oocytes inseminated with 1×10⁵ and 1×10⁴ sperm/ml, respectively. With the low sperm concentration 2/4 of those which were normally fertilized developed to healthy blastocysts. These studies suggest that the zona-drilling technique as described can be performed without apparent harm to oocytes and generate normal embryos. Micromanipulation by zona drilling should be explored further as a potential treatment mode for cases of oligol asthenospermic infertility.     

Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin

OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.     

In vitro maturation, fertilization, and development of human germinal vesicle oocytes collected from stimulated cycles

Objective: To evaluate whether germinal vesicle (GV) oocytes from stimulated cycles can be a source of embryos.

Design: In vitro model study.

Setting: Specialized laboratory of women’s clinic.

Patient(s): Women in whom oocytes were retrieved at the GV stage.

Intervention(s): After culture of GV oocytes in the modified TLP medium with human follicular fluid, oocytes that reached the metaphase II stage underwent ICSI. Potential of fertilization and subsequent cleavage of in vitro-matured oocytes was compared with that of an in vivo-matured control.

Main Outcome Measure(s): Maturation rate, rate of pronuclei formation, and developmental activity.

Result(s): The maturation rate of GV oocytes from follicles primed with gonadotropin was not affected by the presence or absence of cumulus. However, the maturation was more synchronous in oocytes with cumulus than in those without cumulus. Proportions of oocytes with two pronuclei and embryos cleaved to the 16-cell stage after ICSI were significantly lower in the oocytes matured in vitro than in the oocytes matured in vivo.

Conclusion(s): Human GV oocytes from stimulated ovaries can be matured, fertilized, and developed in vitro. Production of embryos from GV oocytes will increase the opportunity for achieving pregnancy.     

兔卵母细胞胞质内单精子注射后二次辅助激活对兔卵母细胞发育能力的影响

目的 观察兔卵母细胞胞质内单精子注射(ICSI)后2次辅助激活对兔卵母细胞发育能力的影响.方法 采集兔卵母细胞500枚,玻璃化冷冻后解冻,行ICSI后培养1 h,将存活的156枚卵母细胞分为10634激活1次组(30枚,加入浓度为5 μmoL/L的钙离子载体10634激活5 min)、氯化锶激活1次组(26枚,加入浓度为10 mmol/L的氯化锶激活20 min)、10634激活2次组(33枚,加入浓度为5μmoL/L的钙离子载体10634激活5 min后培养30 min,采用同样方法再次激活)、氯化锶激活2次组(28枚,加入浓度为10 mmol/L的氯化锶激活20 min后培养30 min,采用同样方法再次激活)和对照组(39枚,未加入任何激活剂)共5组,观察各组卵母细胞的受精率、卵裂率和囊胚形成率.结果 各组兔卵母细胞的受精率、卵裂率和囊胚形成率,氯化锶激活1次组分别为54%、27%和8%,均高于10634激活1次组(分别为33%、17%和3%)和对照组(分别为33%、18%和3%),但差异均无统计学意义(P>0.05);10634激活2次组分别为82%、55%和15%,均高于10634激活1次组,差异均有统计学意义(P<0.05);氯化锶激活2次组的受精率(61%)高于氯化锶激活1次组,卵裂率和囊胚形成率(分别为21%和7%)均低于氯化锶激活1次组,但差异均无统计学意义(P>0.05);10634激活2次组的受精率、卵裂率和囊胚形成率均高于氯化锶激活2次组,差异均有统计学意义(P<0.05).结论 冻融的兔卵母细胞ICSI后行2次辅助激活,可能会提高卵母细胞的受精率和胚胎早期的发育能力,但是激活后的卵母细胞分裂速度较快.     

Passive immunization with antisperm monoclonal antibody ZAP-7 increases preimplantation embryo mortality in the mouse

Objective: To evaluate the effect of VIC-1 and ZAP-7 antihuman sperm monoclonal antibodies on in vivo fertility in the mouse.

Design: A randomized blinded study using a mouse model.

Setting: University-based laboratory.

Animals: B6CBAF1 mice (n = 6 per experimental group).

Intervention(s): Antisperm antibodies were administered intravaginally to female mice before mating. Control mice received no treatment, saline, or nonspecific antibodies. Number and viability of preimplantation embryos were determined by microscopic observation. Mouse sperm, oocytes, and normal preimplantation embryos were used in indirect immunofluorescence assays with antisperm antibodies. The effect of antibody treatment on sperm motility and vitality was evaluated.

Main Outcome Measure(s): Antigen expression, sperm motility and vitality, number and viability of embryos.

Result(s): ZAP-7 antibody recognizes a sperm antigen expressed in zygotes and early preimplantation embryos. Passive immunization with ZAP-7 increases embryo mortality significantly (more than 40% above controls). Passive immunization with VIC-1 has no deleterious effect.

Conclusion(s): ZAP-7 monoclonal antibody disrupts fertilization and embryogenesis in the mouse.     

Eight-hydroxy-2'-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program

Objective: To determine the effects of oxidative stress on the quality of oocytes and embryos, 8-hydroxy-2′-deoxyguanosine (8-OHdG) in granulosa cells was quantitatively studied during an in vitro fertilization and embryo transfer (IVF-ET) program.

Design: Immunocytochemical staining of 8-OHdG in granulosa cells was quantitatively estimated using a charge-coupled device camera and analyzed using the National Institute of Health Image (NIH Image) freeware on a computer.

Setting: Obstetrics and gynecology department in a university hospital.

Patient(s): Ninety-six infertile couples undergoing IVF-ET treatment and intracytoplasmic sperm injection (IVF, N = 72; intracytoplasmic sperm injection, N = 24).

Intervention(s): Oocytes, granulosa cells, and follicular fluids were collected 35–36 hours after the administration of hCG.

Main Outcome Measure(s): 8-OHdG indices were obtained for mural [8-OHdG index (m)] and cumulus [8-OHdG index (c)] granulosa cells.

Result(s): A negative correlation between the fertilization rate and both 8-OHdG indices (c and m) was found. The rate of production of good embryos also showed a negative correlation with the 8-OHdG index (m) and the 8-OHdG index (c). Negative correlations between the 8-OHdG index (c) and E₂ levels in follicular fluid were observed. Endometriosis patients showed a higher 8-OHdG index (c) than did patients with other infertility causes, such as tubal, male factor, and unknown.

Conclusion(s): Oxidative stress in granulosa cells lowered fertilization rates and subsequently led to a decrease in the quality of embryos. The quality of oocytes for endometriosis patients was impaired by the presence of 8-OHdG. This might be one causative factor in infertility in endometriosis patients.     

Pregnancy after immature oocyte donation and intracytoplasmic sperm injection

Objective: To report a case of pregnancy from in vitro-matured primary oocytes fertilized by ICSI. The pregnancy occured in a woman who was in an oocyte donation program; the woman's husband had normal sperm parameters.

Design: Case report.

Setting: Private general hospital affiliated with a university hospital.

Patient(s): A recipient with premature ovarian failure, a recipient's husband with normal sperm, and a pregnant woman who donated her oocytes.

Intervention(s): Aspiration of immature oocytes during cesarean section, in vitro culture for maturation, ICSI of matured oocytes, coculture of fertilized oocytes.

Main Outcome Measure(s): Fertilization of oocytes by ICSI, and cleavage of embryos by Vero cell coculture.

Result(s): Two of seven immature oocytes became metaphase II oocytes, and both were fertilized by ICSI. The two zygotes were cocultured on Vero cells to become grade 1 two-cell embryos. Pregnancy was obtained after transfer.

Conclusion(s): More studies are necessary to clarify whether ICSI can increase the fertilization rate of in vitro-matured primary oocytes, and to clarify the role of coculture in fertilization.     

Intracytoplasmic sperm injection could minimize the incidence of prematurely condensed human sperm chromosomes

Objective: To compare the fertilization and prematurely condensed human sperm chromosomes (PCCs) rates between two intracytoplasmic sperm injection (ICSI) techniques.

Design: A retrospective study.

Setting: The data were obtained from the University of British Columbia in vitro fertilization (IVF) laboratory.

Patient(s): ICSI cycles (n = 105) were performed for couples suffering from severe male-factor infertility and dysfunction of fertilization.

Intervention(s): Two types of ICSI techniques were used for ICSI procedures.

Main Outcome Measure(s): Fertilization and pregnancy rates in group B using the improved ICSI technique were compared with those of group A using the standard ICSI technique. Unfertilized oocytes from the two groups were studied with cytogenetic methods.

Result(s): Oocyte damage dropped from 14.8% in group A to 5.3% in group B. Normal fertilization for each group was 57.3% and 88.4%, respectively (P<.05). Pregnancy rate per egg retrieval was 15.6% in group A and 27.4% in group B (P<.05). PCCs occurred in 19.4% of unfertilized oocytes in group A and did not occur in group B.

Conclusion(s): This study indicates that ICSI not only yields high fertilization rates, but also minimizes the incidence of PCCs. It may be directly related to two crucial steps (immobilization of sperm and aspiration of oocyte cytoplasm) used in ICSI procedures. This study also suggests that it is possible to overcome one cause of IVF failure resulting from the formation of PCCs by using the improved ICSI technique.     

Interaction of Interceed oxidized regenerated cellulose with macrophages: A potential mechanism by which Interceed may prevent adhesions

OBJECTIVE: The objective of the study was to determine whether Interceed oxidized regenerated cellulose (Johnson & Johnson Medical, Arlington, Tex.), because of its polyanionic nature, may compete for the macrophage scavenger receptor. STUDY DESIGN: RAW macrophages were incubated with Interceed oxidized regenerated cellulose and known scavenger receptor ligands. The production of interleukin-1β by mouse peritoneal macrophages was measured in the presence of Interceed cellulose. RESULTS: When macrophages were incubated with Interceed cellulose, increasing concentrations inhibited the uptake of fluorescent acetyl low-density lipoprotein. In the presence of Interceed cellulose there was a decrease in the production of interleukin-1β by mouse macrophages. CONCLUSION: These results suggest that the interaction of Interceed oxidized regenerated cellulose with macrophages with scavenger receptors may result in a decreased secretion of matrix components, inflammatory mediators, and cellular growth factors. Thus Interceed cellulose may function as a biologic barrier in preventing adhesions.(Am J Obstet Gynecol 1997;177:21)     

Numerical dose-compensated in vitro fertilization inseminations yield high fertilization and pregnancy rates

Objective: To evaluate in cases with morphologically abnormal sperm whether fertilization and pregnancy rates are increased by normalizing the number of sperm inseminated and whether biomarkers can identify cases of reduced or failed fertilization.

Design: Prospective studies of sperm morphology and function.

Setting: University hospital assisted human reproduction program.

Patient(s): Partners of 308 women undergoing IVF.

Intervention(s): Motile sperm populations were assessed for sperm head morphology, for surface receptors for mannose and progesterone binding, and the ability to undergo a free mannose-induced acrosome reaction. Zinc in seminal plasma was determined by atomic absorption spectroscopy.

Main Outcome Measure(s): Sperm morphology was associated with fertilization and clinical pregnancy rates. Biomarker analyses were correlated with fertilization rates using Kruskal-Wallis tests, χ² tests, and Spearman rank order correlations.

Result(s): Fertilization and pregnancy rates after numerical dose compensation inseminations were indistinguishable between men with differing percentages of normal sperm. Biomarker deficits were identified irrespective of sperm head morphology in 96% of cases of reduced or failed fertilization.

Conclusion(s): Fertilization and pregnancy rates in cases of abnormal morphology are optimized by inseminating at least 25,000 sperm/mL with normal acrosomes. Reduced or failed fertilization can be predicted by testing for molecular deficits in mannose receptor expression and mannose-stimulated acrosome loss.     

Enhancing in vitro fertilization of mouse oocytes by partial zona pellucida digestion

This work was undertaken in order to evaluate the effect of partial zona digestion on fertilization in vitro of mouse oocytes and assess zona surface changes induced by the procedure. Three hundred forty-six oocytes allocated for treatment were exposed to Ham's F-10 medium supplemented with 0.5% Pronase for either 3 min (188 oocytes) or 5 min (158 oocytes); 324 oocytes served as controls. Oocyte losses incurred as a result of the procedure were small (15 oocytes; 4.3%). Control and Pronase-treated oocytes were each divided into four subgroups and inseminated with 5 ×10 ⁵,5 ×10 ⁴,5 ×10 ³,or 5 ×10 ² sperm cells/ml. Fertilization was assessed 8 hr following insemination by the appearance of two pronuclei and development to the two- to four-cell stage the following day. The morphology of the zona pellucida following Pronase treatment was assessed by phase-contrast and scanning electron (SEM) microscopies performed immediately after treatment. Fertilization rate of control oocytes was 80% at a sperm concentration of 500,000/ml and gradually declined to ~30% at 500 cells/ml. In contrast, treated oocytes inseminated with 500 sperm cells/ml demonstrated a normal rate of fertilization. At this low sperm concentration the longer Pronase treatment was significantly (P < 0.05) more efficient in enhancing fertilization (69 and 88% for 3 and 5 min of Pronase treatment, respectively). Polyspermic fertilization was not observed in any of the subgroups. Phase-contrast microscopic examination of oocytes at the time of Pronase treatment showed an initial swelling of the zona pellucida for 30–60 sec with a time-dependent increase in its transparency. SEM demonstrated that the fine meshlike structure of the outer surface of zona pellucida digested away, leaving a smoother surface. These morphologic changes were not associated with a diminution in sperm binding or penetration. This work demonstrates that partial zona digestion, which causes uniform dissolution of the zona pellucida and reduction of its thickness, is simple and safe. The procedure significantly increased fertilization efficiency at very low sperm concentrations and could, by itself or in conjunction with other methodologies, improve the reproductive capacity of men producing sperm with a reduced penetrating ability.     

冻融后小鼠卵母细胞培养时间对ICSI结果的影响及受精失败辅助激活的结果

目的:研究冻存后的小鼠成熟卵母细胞复苏后培养不同时间对ICSI结果的影响,观察Ca2+载体霉素联合嘌呤霉素对ICSI受精失败的卵母细胞补救激活的有效性。方法:采用慢冻-快融程序化冷冻方法冻融小鼠成熟卵母细胞,复苏后卵母细胞分别培养不同时间(1h、2h、3h、4h、5h)后行ICSI,比较其受精和胚胎发育情况。冻融后受精失败的卵母细胞分为两组(A组:辅助激活,B组:不采取辅助激活),另外获取ICSI受精失败的新鲜卵母细胞(C组)采用和A组同样方法辅助激活,观察3组激活效果。结果:复苏的卵母细胞ICSI前培养3~4h后正常受精率、囊胚形成率明显高于培养1h、2h和5h实验组;A组激活率与B组相比,有明显差异(30.4%vs 6.7%,P<0.05);A组激活率和2PN2PB比例明显低于C组(30.4%vs 75.6%,P<0.05;21.4%vs 47.1%,P<0.05);A、C两组之间1PN2PB比例相比无统计学差异。结论:复苏的成熟卵母细胞ICSI前培养3~4 h后有助于卵母细胞结构的恢复,提高正常受精率和后期发育潜能。辅助激活在一定程度上可以挽救ICSI受精失败的卵母细胞;由于冻融损伤,复苏后ICSI受精失败的卵母细胞被辅助激活的能力明显下降。     

Microinjection of human oocytes: a technique for severe oligoasthenoteratozoospermia.

OBJECTIVE: To determine outcome after microinjection with very poor quality sperm and after failed fertilization. DESIGN: Group 1, fresh oocytes from patients with very low sperm density and motility on the day of oocyte recovery; Group 2, 1-day-old oocytes that failed to fertilize. SETTING: All material was obtained from the National University Hospital. PATIENTS: One hundred and thirty-one from group 1; 35 from group 2. INTERVENTIONS: Sperm was injected subzonally or directly into the ooplasm. MAIN OUTCOME MEASURE: Normal and abnormal fertilization and pregnancy. RESULTS: Subzonal transfer was done on 771 oocytes in group 1 and 188 oocytes in group 2. Multiple sperm were transferred [mean of 6.6 for group 1 and 7.3 for group 2]. Monospermic fertilization occurred in 16.6% and 14.9%, respectively. Polyspermy and parthenogenetic activation were low at 2.3% and 2.8%, respectively. Five pregnancies were obtained, but only one delivered. Ooplasmic injection (single sperm heads) was done in 38 oocytes from three patients with extremely severe oligozoospermia; only four two-pronuclear zygotes were obtained and replaced into two patients, without any resulting pregnancy. CONCLUSIONS: Subzonal transfer may be a viable technique for patients with severe sperm problems.     

Outcome of In Vitro Fertilization and Intracytoplasmic Injection of Epididymal and Testicular Sperm Extracted from Patients with Obstructive and Nonobstructive Azoospermia

Objective: To evaluate IVF outcome after epididymal and testicular sperm retrieval in patients with obstructive or nonobstructive azoospermia.

Design: Retrospective clinical analysis.

Setting: Public university–affiliated IVF unit.

Patient(s): One hundred twenty-three azoospermic patients (178 cycles).

Intervention(s): Sixty-three patients (103 cycles) with obstructive azoospermia (group 1) underwent either epididymal or testicular sperm retrieval, and 60 patients (75 cycles) with nonobstructive azoospermia (group 2) underwent testicular sperm retrieval combined with IVF treatment. Mature oocytes were fertilized using intracytoplasmic sperm injection. After sperm preparation, supernumerary spermatozoa were cryopreserved.

Main Outcome Measure(s): Oocyte fertilization rate and clinical pregnancy rate (PR).

Result(s): The oocyte fertilization rate was 48.4% (534/1,104) in group 1 and 41.5% (312/751) in group 2 (not significant [NS] difference). A total of 100 cycles (97.1%) and 62 cycles (82.7%) in the obstructive and nonobstructive groups, respectively, had embryos for replacement (NS difference). The clinical PRs per ET cycle were 24% (24/100) and 17.7% (11/62) in the two groups, respectively. Oocyte fertilization rates, when fresh (46.4%) or frozen-thawed (41.8%) spermatozoa were used, were not significantly different in the two groups. The PR when fresh sperm were used was 23.6% (30/127), versus 14.3% (5/35) when frozen sperm were used (NS difference). The PR for women aged ≤35 years was similar to that for women >35 years of age (20.7% or 29/140 and 18.2% or 4/25, respectively).

Conclusion(s): Epididymal and testicular sperm obtained in azoospermic patients can fertilize oocytes successfully and may lead to high fertilization rates and PRs. Freezing of these spermatozoa does not reduce the outcome of treatment significantly.     

Clinical significance of human sperm-zona pellucida binding

Objective: To assess the relationship between sperm morphology and motion parameters and sperm-zona pellucida (ZP) binding capacity under hemizona assay (HZA) conditions and to determine the discriminatory power of the HZA for the prediction of in vitro sperm fertilizing ability.

Design: Prospectively designed study.

Setting: Academic tertiary centers.

Patient(s): One hundred ninety-six couples undergoing IVF therapy participated in this study.

Intervention(s): Hemizona assay and IVF results were determined for each couple.

Main Outcome Measure(s): Computerized sperm motion analysis, sperm morphology (strict criteria), and HZA results were correlated with fertilization outcome.

Result(s): Among sperm parameters from the original ejaculates, morphology was the best predictor of sperm-ZP binding ability; hyperactivated motility was the best predictor of HZA results after swim-up separation of the motile sperm fractions. The HZA index provided the highest discriminatory power for fertilization success/failure, with an overall accuracy of 86%.

Conclusion(s): Sperm morphology and hyperactivated motility showed a high correlation with the capacity of sperm to achieve tight binding to the ZP. The excellent positive and negative predictive values of the HZA for fertilization outcome provide additional support for the use of this functional bioassay in the decision-making process within the assisted reproduction setting.     

A human sperm-zona pellucida binding test using oocytes that failed to fertilize in vitro

A test for human sperm binding to the zona pellucida (ZP) was developed using oocytes which failed to fertilize in vitro. Heterospermic insemination with equal numbers of test and fertile donor sperm differentially labeled with fluorescein isothiocyanate or tetra-methylrhodamine B isothiocyanate controlled for variability in ZP-sperm binding capacity. The number of sperm bound to the ZP was independent of previous sperm binding in in vitro fertilization (IVF), preservation of the ZP in salt solution, and fluorochrome labeling but increased linearly with time and sperm concentration. Sperm from men who had one or more failed attempts at IVF with no or few oocytes fertilized usually displayed very low ZP binding ratios of test to normal sperm. This test may predict the ability of sperm to fertilize human oocytes in vitro and should be useful in studies of human gamete interaction.