Teri Ord

Micromanipulation of sperm by a laser generated optical trap

The force generated by the radiation pressure of a low power laser beam induces an optical trap which may be used to manipulate sperm. We studied the effect of the optical trap on sperm motility. A Nd:YAG laser beam was coupled to a conventional microscope and focused into the viewing plane by the objective lens. Sperm were caught in the trap and manipulated by a joy stick controlled motorized stage. After different exposure periods, the velocity and patterns were analysed by a computerized image processor. There were minor changes in sperm velocity when exposed to the trap for 30 seconds or less. A gradual decrease in the mean linear velocity was observed after 45 seconds of exposure. This optical micromanipulator may also be useful for studying the force generated by a single spermatozoa and evaluating the influence of drugs on motility.

Force generated by human sperm correlated to velocity and determined using a laser generated optical trap

The development of the single beam gradient force optical trap has made it possible to manipulate cells solely by laser light. A continuous wave Nd:YAG (1.06 microns) laser beam was directed into a conventional microscope and focused onto the viewing plane by the objective lens. The laser beam power at which human sperm were released from the trap was measured and correlated to the sperms linear velocity before trapping. The mean trapping power readings for slow, medium, and fast motile sperm were 57, 73, and 84 mW, respectively. The analysis of measurements over the total population demonstrated that zig-zag motile sperm had significantly higher mean power readings when compared with straight motile sperm with similar mean linear velocities. In two cases, specimens required significantly less trapping power when the measurements were repeated 24 hours later.

Oocyte donation and gamete intrafallopian transfer in premature ovarian failure

Gamete intrafallopian transfer (GIFT) was performed in eight patients with premature ovarian failure (POF), using donated oocytes. The steroid replacement protocol consisted of the administration of increasing dosages of 17β-estradiol (E2) and progesterone (P). Hormonal replacement was maintained until day 100 of gestation. All patients underwent an evaluation cycle in which serum levels of E2 and P were monitored and an endometrial biopsy was performed on day 21 or 22. All cases of GIFT were performed between days 12 and 15. Six clinical pregnancies were achieved in eight cycles (75% success rate). Three patients delivered and three are in their second or third trimester. No ectopics or miscarriages occurred. These results offer a promising approach for the establishment of fertility in agonadal patients.

Tubal embryo transfer as a treatment for infertility due to male factor

Transvaginal follicular aspiration (TVA) with ultrasonically guided needles allows the transfer of in vitro generated embryos to the fallopian tubes (TET), performing only one surgical procedure in the process. Up to now, this approach has been used to treat 16 couples with infertility due to severe male factor. Follicular development was induced with a combination of clomiphene citrate and human menopausal gonadotropin (hMG) or follicle-stimulating hormone and hMG. Follicles were aspirated by TVA 36 hours after an injection of human chorionic gonadotropin 10,000 IU intramuscularly. A total of 169 oocytes were recovered (10.5 +/- 6.9 X +/- SD) from the 16 patients. There was failure of fertilization in 6 cases. In the remaining 10, a TET was performed 44 to 50 hours after TVA, utilizing embryos at the pronuclear stage. Six pregnancies resulted from the 10 transfers. This technique combines the advantages of proof of fertilization with a more adequate tubal embryo development and entrance to the uterine cavity that may determine and increase chance of implantation.

Movement characteristics of human epididymal sperm used for fertilization of human oocytes in vitro

Study Objective To develop mathematical models using kinematic parameters from Computer-Aided Sperm Analysis (CASA) that predict the fertilization rate of sperm recovered from the caput epididymidis and to test the hypothesis that fertilization was enhanced by the presence of specific sperm subpopulations in the inseminate. Setting In vitro fertilization (IVF) program. Patients Thirteen patients with congenital absence of the vas deferens provided epididymal sperm for IVF as well as for CASA. Results The mathematical model that was most predictive of fertilization rates included kinematic parameters of the epididymal aspirate (percent motility), the inseminate used for IVF (curvilinear velocity [VCL]), and the change in sperm movement after in vitro processing by the mini-Percoll technique (difference in amplitude of lateral head displacement [ALH]). Multivariate cluster analysis revealed that inseminates that resulted in higher fertilization rates had subpopulations of sperm that were characterized by high VCL and high mean angular displacement, as well as a greater change in ALH after processing. Conclusion In vitro fertilization with epididymal sperm was more likely to succeed when the sperm population that was initially aspirated had a higher proportion of motile cells and when these sperm were capable of capacitation in vitro as indicated by the appearance of sperm subpopulations with motility that resembled hyperactivation.

Synthetic serum substitute (SSS): a globulin-enriched protein supplement for human embryo culture.

ObjectiveThe purpose of the present study was to evaluate whether an IVF protein supplement prepared from human serum albumin (HSA) and human globulins would retain performance characteristics equivalent to those reported for the commercial plasma expanders, Plasmatein (Alpha Therapeutics, Los Angeles, California) and Plasmanate (Cutter Biological, Miles Inc., Elkhart, Indiana).MethodsPronuclear-stage human embryos were randomly divided and cultured in human tubal fluid medium (HTF) supplemented with either HSA (5 mg/mL) or Plasmatein (10%, v/v; 5 mg/ml as a means of indirectly assessing the effect α- and Β- globulins have on embryonic development. Those results coupled with the known composition characteristics of Plasmatein were used as the starting basis to formulate test lots of synthetic serum substitute (SSS).ResultsSignificantly (P<0.05) more of the human embryos cultured in Plasmatein supplemented medium reached the four-cell or greater stage by 40 hr postinsemination than a comparable group cultured in HSA alone. Lot 1 of SSS, formulated with HSA (84% of total protein) and human globulins (16% of total protein) and an aqueous lipoprotein fraction derived from human plasma (Excyte IV; Miles Diagnostics, Kankakee, Illinois), produced accelerated early embryonic growth relative to control murine embryos grown in the presence of Plasmatein, however, the percentage of the embryos reaching the hatched blastocyst stage was decreased (45 vs 100%). Human embryos from seven patients, randomized to HTF medium supplemented with Plasmatein or lot 1 of SSS, showed equivalent growth at 36– 40 hr postinsemination. A microprecipitate developed in media supplemented with lot 1 after several days of culture. The Excyte IV concentration was reduced and, ultimately, eliminated from the subsequent and final prototype lots of SSS. Murine embryos grown in the presence of lipoprotein free SSS showed significantly accelerated (P<0.01) growth at 17 hr postthaw compared to Plasmatein and all embryos progressed to hatching by 41 hr. Human embryos, randomized to either Plasmatein or lot 3 of SSS, showed significantly accelerated growth (P<0.01) when scored at 38 hr following insemination.ConclusionSynthetic serum substitute provides a convient, standardized means of adding protein to media used in assisted reproductive technology (ART) procedures.

Can severe male factor infertility be treated without micromanipulation

OBJECTIVE To present data on IVF in severe male factor infertility using modified IVF laboratory methods and to compare them with the reported data of gamete micromanipulation. DESIGN Retrospective, not randomized. SETTING University of California, Irvine, Center for Reproductive Health. PATIENTS Seventy-one patients with severe oligoasthenozoospermia defined as total motile count in the pretreatment sample of < 5 x 10(6). Two groups were identified, group I with total motile count between 1.5 and 5 x 10(6) and group II with total motile count < 1.5 x 10(6). INTERVENTIONS Treatment of the semen samples with mini-Percoll and the modification of standard IVF techniques. These modifications include insemination with larger numbers of sperm, the use of culture tubes for insemination of oocytes, and pooling oocytes together in one or more culture tubes. MAIN OUTCOME MEASURES Fertilization and pregnancy rates (PRs). Additionally, the number of patients achieving cryopreservation and patients with previous failure to fertilize are specified. RESULTS Eighty percent of the patients achieved fertilization with an overall rate of 38% per oocyte. Fifty-three ETs were performed and 23 clinical pregnancies (43% per transfer and 35% per cycle) were achieved. Fifty percent of the patients had excess embryos for cryopreservation. In group I the fertilization rate was 54% with a PR of 56% per transfer and 48% per cycle. In group II the fertilization rate was 25% with a PR per transfer of 32% and 24% per cycle. Of 28 patients who had previous failure of fertilization, 25 fertilized and 8 pregnancies were established. CONCLUSIONS These data demonstrate that by the modification of standard laboratory methods for IVF, monospermic fertilization, cleavage, and a high clinical PR can be achieved in cases of severe male factor infertility without having to resort to micromanipulation.

Successful pregnancy outcome after cryopreservation of all fresh embryos with subsequent transfer into an unstimulated cycle

OBJECTIVE To assess the pregnancy outcome of freezing and storing all fresh embryos produced in a stimulated IVF cycle and replacing them in a subsequent nongonadotropin-stimulated cycle. DESIGN Retrospective study. SETTING University-associated assisted reproductive technology program. PATIENTS We studied 36 patients (age range 23 to 44 years) who underwent cryopreservation of all fresh embryos in a controlled ovarian hyperstimulation (COH) cycle because of either the risk of severe ovarian hyperstimulation (24 patients, group 1) or the presence of an endometrial lining < 8 mm in thickness (12 patients, group 2). Five hundred fifty-five embryos were generated for replacement in 63 cycles. All embryos were cryopreserved in 1.5 M propanediol at the pronuclear or two-cell stage, and 264 embryos subsequently were transferred into a hormone replacement cycle (70%) or natural ovulatory cycle (30%). The average number of embryos transferred per patient was 4.2. RESULTS Twenty-one clinical pregnancies were achieved, giving a pregnancy rate (PR) of 58.3% per patient (33.3% per cycle). The live birth rate was 50% per patient (28.6% per cycle). The implantation rate was 9.1%. Groups 1 and 2 had a similar PR per patient (58.3%). With 208 cryopreserved embryos remaining and considering the 33.3% PR per cycle, we expect the overall extrapolated PR to be 63.9%. CONCLUSIONS This is the first series showing that freezing and storing all fresh embryos produced in a stimulated IVF cycle and replacing them in a subsequent nongonadotropin-stimulated cycle results in successful PRs. These results underlie the importance of a successful cryopreservation program in IVF and could be a possible approach to overcoming the alleged adverse effects of COH on the endometrium, thereby improving the chances of pregnancy when numerous embryos are obtained simultaneously.

Embryo implantation rates in oocyte donation: a prospective comparison of tubal versus uterine transfers *

OBJECTIVE To compare pregnancy and implantation rates in tubal and uterine transfers during a hormonal replacement cycle in an oocyte donation program. DESIGN Prospective randomized. PATIENTS Forty-two consecutive patients who entered an oocyte donation program. INTERVENTIONS Twenty-two patients were assigned for uterine transfer and 20 for tubal embryo transfer (ET). RESULTS Twenty-three pregnancies were achieved, 12 (54.5%) after uterine transfers and 11 (57.9%) after tubal transfers. Implantation rates in both groups are not significantly different (17.4% uterine transfers versus 21.5% tubal ETs). CONCLUSIONS Our results suggest that in hormonal replacement cycles (uniform endometrial stimulation) there is no advantage in transferring embryos to the fallopian tube. Furthermore, embryo quality and endometrial receptivity appear to be significantly more important than the time of entrance of an embryo to the uterine cavity in determining its chances of implantation.

Correlation between epididymal length and fertilization rate in men with congenital absence of the vas deferens

OBJECTIVE To investigate whether the variable length of the epididymides in men with congenital absence of the vas deferens might have a correlation with IVF and pregnancy rate results. DESIGN Microsurgical retrieval of epididymal sperm from men with congenital absence of the vas deferens and their use for IVF. SETTING Center for Reproductive Health, University of California, Irvine, California. PATIENTS One hundred eight men with confirmed diagnosis of congenital absence of the vas deferens enrolled in the microsurgical epididymal sperm aspiration and IVF program. INTERVENTIONS Measurement in centimeters of the epididymal length at the time of the sperm aspiration procedure. MAIN OUTCOME MEASURE Rates of fertilization and pregnancy according to the epididymal length. RESULTS Three groups were identified: group I (n = 29), epididymal length between 0.5 and 1.9 cm; group II (n = 66), length between 2.0 and 4.0 cm; and group III (n = 13), length in excess of 4.0 cm. Although the aspiration site was the proximal caput for each case, patients of group III had the highest fertilization and pregnancy rate (24% and 43%, respectively). Patients with the shortest epididymis (group I) had the worst IVF outcome (fertilization rate 7% and pregnancy rate 7%) whereas in group II the fertilization rate was 13% and the pregnancy rate was 18%. CONCLUSION This study demonstrates that epididymal sperm from men with congenital absence of the vas deferens having a longer epididymis have a better IVF rate. A long epididymis can allow [1] the arrival of more frequent waves of fresh sperm whereas in a short epididymis the system is completely congested and occupied by old and senescent sperm, [2] less obstructive damages, and [3] a back flow of biochemical factors produced in the more distal segments that could ultimately enhance the fertilization capacity of proximal epididymal sperm.