The absence of outer dynein arms in the sperm flagellum induces an abnormal movement pattern associated with male infertility. These spermatozoa can decondense in zona-free hamster oocytes but result in a very low fertilization rate in in vitro fertilization. We hypothesized that subzonal insemination could help achieve fertilization and pregnancy. A randomized prospective trial (five couples, five cycles) comparing subzonal insemination (n = 31 oocytes) and routine IVF (n = 23 oocytes) was carried out. Oocytes were microinjected with 8.5 +/- 3.6 spermatozoa. In a second series (nine cycles), all the oocytes were microinjected with 10.5 +/- 4.3 spermatozoa. In the randomized series, the fertilization rate was 16.1% without polyploidy, whereas no fertilization was obtained after control IVF insemination. In the second series involving nine couples, six of whom were included in the first series, the fertilization rate increased to 57.8% with a 27.8% polyspermic rate. Eighty-eight per cent of the zygotes cleaved normally (29 out of 33). A total of 11 embryo transfers resulted in three pregnancies, one of which terminated one month later, a second being ongoing and the third delivering a healthy girl. A 21.4% pregnancy rate per cycle, with a 37.5% pregnancy rate per couple, justifies the use of subzonal insemination to treat this particular flagellar dyskinesia.
Flagellar dyskinesia is characterized by abnormal sperm movement parameters and a negative sperm mucus penetration test. It is associated with structural pathologies of the axonemal complex (lack of outer dynein arms), of the periaxonemal complex (sliding spermatozoa and periaxonemal dyskinesia), or of both structures (short flagella). Even during in vitro fertilization, dyskinesia prevents the spermatozoon from getting through the egg vestment. However, in some cases, fertilization has been achieved using subzonal insemination. Flagellar dyskinesia is therefore an interesting model for investigating the role of sperm movement in the fusion process between the spermatozoon and the oolemma. Thirty-one patients requiring assisted fertilization were included in the study. Fifteen had spermatozoa in which the flagellum lacked outer dynein arms, 11 had anomalies of the periaxonemal complex (five with sliding spermatozoa and six with periaxonemal dyskinesia) and five had spermatozoa with short flagella. Seven men who produced spermatozoa with normal movement were selected as controls. Movement was evaluated using a computer-assisted analyser, and penetration was assessed using zona-free hamster eggs. At 37 degrees C, in semen, the dyskinetic spermatozoa had reduced straight line and curvilinear velocity and lateral head displacement compared with controls (P < 0.01). In the Percoll-selected sperm suspension, the only difference was that spermatozoa with periaxonemal anomalies maintained a narrow lateral head displacement compared with the controls (P < 0.001). After 3 h of incubation at 37 degrees C, the lateral head displacement of dyskinetic spermatozoa had not changed, while that of the controls showed a significant increase (4.5 to 5.6 microns; P < 0.05). The results from the sperm penetration assay for the spermatozoa lacking outer dynein arms were lower than those of the controls (47% versus 77%; P < 0.05) and the results for sliding spermatozoa and spermatozoa with periaxonemal dyskinesia were even lower (25% and 34%, respectively; P < 0.01). The fertilization rates after subzonal insemination were 46.5% for spermatozoa lacking outer dynein arms, 36.1% for spermatozoa with short flagella, 24.8% for sliding spermatozoa and 17.3% for spermatozoa with periaxonemal dyskinesia. There was a significant correlation between the curvilinear velocity of the Percoll-selected sperm suspensions and their fertilization rates after subzonal insemination (r = 0.5; P < 0.05) and their sperm penetration assays (r = 0.7; P < 0.001). The data provide evidence that sperm velocity is correlated with the ability to fuse with the oolemma.
Subzonal insemination has been proposed to achieve fertilization in cases where standard in vitro fertilization has failed. We present the results of chromosome analysis of oocytes after subzonal insemination. Our data suggest that the main cause (76 per cent) of the absence of cleavage after subzonal insemination is the total absence of sperm nucleus evolution of the injected spermatozoa. Our results also suggest that spermatozoa chromatin development is normal after subzonal insemination. Aneuploidy does not seem to be increased in zygotes after subzonal insemination. However, polyploidy was often more important than predicted by the observation of pronuclei (PN). Pronucleus development might be asynchronous and can appear earlier or later than after standard IVF. The cytogenetic risk after subzonal insemination might therefore be triploidy (if a triploid egg is transferred, because only 2 PN were seen) rather than aneuploidy or structural abnormalities.
The cytometry of 545 oocytes was evaluated during subzonal insemination (SUZI; 85 attempts), on day 0 (egg retrieval and SUZI), day 1 and day 2 (embryo transfer). On day 0, the egg and oolemma diameters (mean ± SD) were 164.0 ± 19.6 urn and 114.2 ± 16.8 u5m respectively. The zona thickness was 17.8 ± 13.4 urn and correlated with the oolemma diameter (r = 0.24, p < 0.001). The fertilisation rate was significantly lower for the smaller oocytes (less than 108 urn diameter) compared with the larger oocytes (over 108 urn) (9.8% vs 21.2% respectively; p < 0.05). There was little variation in oocyte diameter according to nuclear status. However, oocyte diameter increased significantly between day 0 and day 1 (p < 0.001) for both fertilised and unfertilised oocytes. Six different indications for SUZI were investigated in detail: three with non-specific (normal and subnormal sperm with in vitro fertilization failure, oligoasthenospermia) and three with specific sperm defects (flagellar dyskinesia, absence of outer dynein arms, antisperm antibodies). Oocytes from the non-specific defect groups had significantly smaller diameters than the others (p < 0.05). The mean fertilisation rate was related to the mean oolemma diameter for the groups with non-specific sperm defects and the group lacking dynein arms (LODA) (r = 0.91, p < 0.05). Eggs from the groups of patients with LODA and those with antisperm antibodies had thicker zona pellucida than others (p < 0.05). These findings suggest that in addition to nuclear criteria of maturity, the growth of oocytes is an important factor for fertilising ability. Insufficient development of the ooplasm may contribute to fertilisation failure, particularly when sperm with functional defects are used. In contrast, a thick zona pellucida may prevent sperm with specific anomalies such as LODA or antisperm antibodies from penetrating into the perivitelline space.
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