Heng-Ru Lin

Comparison between a two-layer discontinuous Percoll gradient and swim-up for sperm preparation on normal and abnormal semen samples

PurposeThis work was to compare the effects of Percoll gradient and swim-up treatments for sperm preparation on the percentage of progressive motility, recovery of motile sperm, removal of debris, percentage of normal forms according to strict criteria, and movement characteristics of sperm using computer-assisted velocity analysis.ResultsIn total, 50 semen samples from 50 patients were tested and divided into two groups: a normal group (n=27) with normal parameters and an abnormal group (n=23) with abnormal parameters. The results in both the normal and the abnormal groups revealed that the sperm concentration in the Percoll samples was significantly greater than that in the swim-up samples. Although the percentage of progressive motility was greater in the swim-up samples than in the Percoll samples, the number of motile sperm, reflecting the percentage of motile sperm recovery, was still greater in the Percoll samples. The debris of semen were equally removed by both methods and the percentage of normal forms was also similar in the samples treated according to these two procedures. Both curvilinear velocity (VCL) and straight-line velocity (VSL) of sperm were significantly greater in the swim-up samples than in the Percoll samples. Sperm from the swim-up procedure also showed a greater mean amplitude of lateral head displacement than that from the Percoll gradient procedure, but the distinction was insignificant.ConclusionThe Percoll gradient technique, by recovering more motile sperm, may be applied to prepare oligospermic samples. The swim-up method may become the standard choice to prepare normal semen which could obtain sufficiently motile sperm, due to its simplicity and recovered sperm with superior motility.

Correlation between sperm morphology using strict criteria in original semen and swim-up inseminate and human in vitro fertilization.

To study the value of sperm morphology using strict criteria in raw semen and in swim-up inseminate of human in vitro fertilization (IVF), 135 cycles of IVF with normal sperm concentration and motility were recruited. At least two mature oocytes were recovered in each cycle. The correlation between the percentages of normal forms and fertilization rates of mature oocytes was analyzed. The results demonstrate that the percentage of normal forms in both the raw semen and swim-up sample of patients with poor fertilization was significantly lower than in those with acceptable fertilization. The percentages of normal forms both in raw semen and in swim-up sample were significantly correlated with fertilization rates in vitro, however, the former seemed to have a better correlation (r = .51 and .19, respectively). Regarding the percentages of normal forms in raw semen, the fertilization rate in patients with normal forms < 4% was 6 +/- 11%, for 4-14% it was 58 +/- 36%, and for > 14% it was 88 +/- 20%. The fertilization rates were significantly different among these three groups of patients. The evaluation of sperm morphology using strict criteria in raw semen before IVF is predictive of fertilization outcome and may also help doctors to choose an optimal method of treatment for patients.

PEPTIDES EXTRACTED FROM VERO CELL CULTURES OVERCOME THE BLASTOCYST BLOCK OF MOUSE EMBRYOS IN A SERUM-FREE MEDIUM

PurposeThe aim of this work was to evaluate the effect of a Vero cell coculture system on the development of mouse embryos.MethodsMouse embryos were randomly divided and cultured in human tubal fluid (HTF) medium with/without Vero cell monolayers, conditioned medium (CM) obtained from Vero cell cultures, and HTF medium supplemented with peptides extracted from CM. The concentrated CM was examined by SDS/PAGE.ResultsThe development of mouse embryos was blocked at the blastocyst stage in pure HTF medium (1.4% hatching at day 5). This “blastocyst block≓ was overcome by coculture with Vero cell monolayers (48.1% hatching at day 5; 1.4 vs 48.1%; P<0.001). CM and the addition of 5% fetal bovine serum (24.1 and 34.9% hatching, respectively, at day 5) were also able to enhance the process of hatching. In the other experiment, the addition of peptides extracted from Vero cell cultures also overcame the blastocyst block (12.5%) compared with pure HTF medium (2.1%) (P<0.05). Electrophoretic separation revealed several classes of polypeptides consistently secreted into CM obtained from Vero cell cultures. Most peptides occurred in the Mrrange between 6.5 kd and 35.9 kd.ConclusionA developmental block (blastocyst block) of mouse embryos in a serum- and protein-free medium (HTF) was discovered in this study. This block was effectively overcome by HTF plus serum and coculture with Vero cell monolayers and also by the peptides extracted from Vero cell-conditioned medium. We speculate that certain factors secreted or converted by Vero cells may be critical in hatching of mouse embryos. Further study of these factors may be helpful in delineating its mechanism.

Effect of the number and depth of embryos transferred and unilateral or bilateral transfer in tubal embryo transfer (TET).

PurposeOur purpose was to evaluate the possible effects of the number of embryos transferred, the depth of embryos placed within the tube(s), and unilateral or bilateral tubal transfer on pregnancy initiation in tubal embryo transfer (TET).MethodsOne hundred eight consecutive TET cycles were analyzed. Oocyte retrievals were carried out by transvaginal ultrasound-guided aspiration of follicles. Forty-eight hours after oocyte retrieval, the developing embryos at the stage of two to four cells were transferred into the fallopian tube(s) by laparoscopy. A maximum of four embryos was transferred to each patient.ResultsThe pregnancy rates were similar among the cycles in which two, three, or four embryos were transferred. In addition, there was no significant difference in the pregnancy rate whether the embryos were deposited >4 cm or between 3 and 4 cm into the tube(s). Although the pregnancy rate was greater in cycles of bilateral tubal transfer, the difference from that of unilateral transfers was not significant.ConclusionOur data indicate that when two to four embryos were transferred and the embryos were placed ⩾3 cm within the tube(s), unilateral or bilateral tubal transfer had little influence on the ultimate success of TET.

Interleukin-1β (IL-1β) is increased in the follicular fluids of patients with premature luteinization

PROBLEM: Most, but not all, studies indicate that premature luteinization correlates with poor pregnancy outcome in in‐vitro fertilization (IVF) programs. It remains unclear whether cytokines (IL‐1β, TNFα), the established immune mediators, play a role in regulation or initiation of an abnormal follicular or embryo development in patients with premature luteinization.

Acrosin Activity of Human Sperm Did not Correlate with IVF

To evaluate the predicting value of sperm acrosin activity in human, the acrosin activity index (AAI) was measured in 95 semen samples from patients participating in an IVF program. All patients had at least two mature oocytes. Of 95 patients, 84 had successful fertilization and 11 failed to fertilize all oocytes in vitro. The numbers of mature oocytes were similar between fertilization and nonfertilization groups. The mean AAI, measured using a commercially available (Accu-Sperm) acrosin activity assay, was greater in the fertilization group than in the nonfertilization group, but the difference was not significant. There was no correlation between AAI and the in vitro fertilization rate of mature oocytes. The relation between AAI and semen parameters also showed no significant difference. It would appear that measurement of AAI inaccurately reflects in vitro fertilizability of human sperm.

Microinjection of human sperm into perivitelline space of hamster eggs: comparison with zona-free hamster egg penetration of human sperm

Micromanipulation of human sperm and oocyte has been utilized to facilitate fertilization of those patients with male factor due to oligoasthenospermia or those patients with repeated fertilization failure in an in vitro fertilization (IVF) program. Before manipulating human gametes, one needs experience with animal models. Our objective was to perform subzonal insertion of human sperm into hamster eggs and to compare the result with that of sperm penetration assay (SPA) using zona-free hamster eggs. Semen samples were obtained from 15 fertile donors with normal semen analysis and the motile sperm were collected by swim-up procedure. Microinjection was performed by injecting a varied number of sperm into the perivitelline space of 222 hamster eggs pretreated with sucrose solution (0.1 M). The rate of damage of eggs during microinjection was 7.2% (16/222). The rates of penetration in the microinjection group were 5.1% (4/79) for 1-5 sperm injected, 10.9% (11/101) for 6-10 sperm injected, and 11.5% (3/26) for 11-15 sperm injected. The average rate of penetration per egg was 8.7% (18/206), and the polyspermic rate was 11.1% (2/18). Simultaneously SPA was performed in each sample of semen as a positive control, and the average rate of penetration of SPA was 51.4% (108/210). The rate of penetration in the microinjection group was significantly smaller (p < .05) than that in the SPA group. Whether the penetration rate and polyspermic rate in a hamster model reflect similar results in human oocyte requires further investigation. However, the hamster egg provides an ideal model to develop a micromanipulation technique for human beings.