Cryopreservation of single human spermatozoa.

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.

Beneficial aspects of co-culture with assisted hatching when applied to multiple-failure in-vitro fertilization patients.

A study was conducted on patients who had attempted and failed previous in-vitro fertilization (IVF) procedures an average of 3.8 times following the application of assisted hatching with conventional culture systems. The aim of this investigation was to determine if addition of co-culture methodologies could reduce embryonic abnormalities and thus improve the prognosis for pregnancy. The study population consisted of 123 patients, subdivided into three patient categories. Previous IVF results from conventional culture were used to evaluate any potential benefits derived from the present co-culture application. Following co-culture, the rate of blastomere development was increased and the rate of fragmentation decreased. An increased rate of blastomere development was most noticeable in the patients aged < or = 39 years with no male factor as well as the intracytoplasmic sperm injection (ICSI) subgroup. Similarly, the rate of fragmentation was significantly reduced in the aforementioned subgroups. The most pronounced impact of co-culture was on pregnancy and implantation rates. The overall clinical and ongoing pregnancy rates were 38% (47/123) and 36% (44/123) respectively. The corresponding implantation rate was 17% (72/ 412) as shown by embryonic cardiac activity. The ongoing pregnancy rates in the < or = 39 years no male factor, > or = 40 years no male factor and ICSI no age limit patient subgroups were 41% (21/51), 30% (8/27) and 33% (15/45) respectively. The results indicate that addition of co-culture to the IVF procedure for poor-prognosis patients may be advisable.

Selective assisted hatching of human embryos.

Overall results of assisted hatching by zona drilling using acidic Tyrode's solution performed during three randomised trials in 330 in vitro fertilisation (IVF) patients are presented. It was demonstrated retrospectively and prospectively that assisted hatching by zona drilling was effective in embryos with thick zonae (> 15 microns). This procedure is called selective assisted hatching. In order to investigate whether the success rate of embryos with thin zonae (< 13 microns) can be improved further, a fourth trial was executed in 40 consenting patients. Embryos with thin zonae were left intact in one group (control), while the outside of zonae of similar embryos were thinned with acidic Tyrode's solution. Results thus far indicate that embryos with thin zonae do not benefit from this technique. Embryonic implantation (fetal heartbeat per embryo) was high (26%-27%) in both arms of the trial, probably as a result of selective zona drilling of low prognosis embryos with thick zonae. A method is presented for quantifying zona hardening in human embryos. The exposure to acidic Tyrode's solution of each embryo was expressed as a function of the duration to pierce the zona and the diameter of the needle. Preliminary findings suggested that embryonic viability is correlated with zona hardening. In order to test the hypothesis that extracellular fragments may affect embryonic viability, small amounts of fragments were removed from embryos during assisted hatching. The pregnancy rate in 36 patients with extracted fragments was relatively high (41%) considering the poor morphology of the embryos involved.

Microsurgical fertilization procedures: the absence of stringent criteria for patient selection.

Subzonal sperm insertion and partial zona dissection were applied in 250 in vitro fertilization cycles in couples (n = 200) with abnormal semen analyses; 61 clinical pregnancies were established (24% per egg retrieval). Patients were selected without using minimal cutoff criteria. The study included patients with 0% normal sperm forms (strict criteria), no motile sperm (but some live cells), and sperm counts which could be assessed only after centrifugation. Patients were categorized into three subsets. Group A (n = 116 cycles) failed to fertilize in a previous cycle. Group B (n = 40) was excluded from IVF due to the severity of sperm profiles, such as a maximum of 2% normal forms. Group C (n = 94) constitutes those patients for whom a standard cycle could possibly result in failure. Monospermic fertilization rates were 18% (A), 19% (B), and 24% (C). The incidences of embryo replacement were 63% (A), 53% (B), and 69% (C). Rates of clinical pregnancy were 22% (A), 23% (B), and 28% (C). The presence of one, two, or three semen abnormalities did not correlate with the outcome of microsurgical fertilization. Twenty-two percent of patients with combined oligoasthenoteratozoospermia became pregnant. Moreover, ongoing pregnancies were established in instances with 0% normal sperm forms and no progressively motile spermatozoa. It is concluded that stringent cutoff criteria may not be necessary when both partial zona dissection and subzonal sperm insertion are performed efficiently.