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.

Micropuncture retrieval of epididymal sperm with in vitro fertilization: importance of in vitro micromanipulation techniques.

OBJECTIVES: To evaluate the importance of in vitro micromanipulation techniques, specifically intracytoplasmic sperm injection (ICSI), for the fertility treatment of men with congenital absence of the vas deferens (CAV) or other unreconstructable male reproductive tract obstruction. METHODS: Results using ICSI during in vitro fertilization (IVF) were compared to previously published results of IVF alone and IVF with other micromanipulation techniques at the same infertility center. Main outcome parameters evaluated were: fertilization rate per oocyte, clinical pregnancy rate, and ongoing pregnancies and deliveries. RESULTS: IVF with ICSI yielded a fertilization rate per oocyte of 140 of 312 (45%) and a clinical pregnancy rate of 14 of 27 (52%) per cycle of sperm and egg retrieval. Ongoing pregnancies or deliveries have occurred for 13 of 27 (48%) cycles with ICSI. These results were better than our previously published results of IVF alone or in conjunction with the micromanipulation techniques of subzonal insertion (SuZI) or partial zona dissection (PZD) that yielded a 119 of 631 (19%; P < 0.0001) fertilization rate, clinical pregnancy rate of 14 of 51 (27%; P < 0.001) and ongoing pregnancy or delivery for 12 of 51 cycles (24%; P < 0.001). CONCLUSIONS: Epididymal sperm retrieval should be performed only when micromanipulation is available in conjunction with IVF to maximize chances of fertilization and subsequent pregnancies. The use of ICSI for epididymal sperm appears to maximize chances of pregnancy for couples with surgically unreconstructable obstructive male infertility.

Plasmanate as a medium supplement for in vitro fertilization.

PURPOSE: The purpose of this study was to evaluate the use of Plasmanate, a protein preparation containing human serum albumin and mixed globulins to autologous preovulatory maternal serum, as an in vitro fertilization (IVF) medium supplement. Plasmanate was used most often in cases involving unexplained infertility, sperm antibodies, and endometriosis or when serum was unavailable. RESULTS: In a retrospective analysis of 1019 consecutive IVF cycles, Plasmanate was used as the protein supplement to the fertilization medium in 28.6% and maternal serum was used in 71.4% of the attempts. Attempting to eliminate the effects of different medium lots and laboratory conditions, 450 matched patient cycles were compared using the two protein supplements. Finally, the effects of Plasmanate versus maternal serum were compared in prospective randomized trial on patients diagnosed with tubal infertility who were attempting IVF for the first time. The clinical pregnancy rate was 34% for the Plasmanate group versus 24% for those using maternal serum in the retrospective investigation. However, this trend was reversed in the prospective trial. CONCLUSION: Although further investigation is necessary, it appears that Plasmanate is an appropriate protein substitute in patient cases where serum is absent or unsuitable.

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.

Subzonal sperm insertion and the frequency of gamete fusion.

The subzonal sperm insertion technique was applied to assess the potential of motile human spermatozoa to form pronuclei. In 184 mature human oocytes, subzonal sperm insertion was used as the primary mode of insemination in cases with abnormal semen analyses. Oocytes (n = 131) that failed to fertilize in vitro in cases with normal semen profiles were also micromanipulated for secondary insemination. The frequency of sperm fusion, expressed as a percentage, was defined as the total number of male pronuclei formed divided by the total number of spermatozoa inserted subzonally. Our results indicate that 37% of spermatozoa from men with normal semen are able to fuse with the oolemma and decondense within the ooplasm, when placed in the perivitelline space of the oocyte. Excluding the oocytes that appeared morphologically abnormal (presence of cytoplasmic inclusions such as refractile bodies within the ooplasm), the frequency of sperm fusion increased to nearly 60%. Moreover, 14% of subzonally inserted spermatozoa from men with abnormal semen analyses demonstrated an ability to form a pronucleus. The incidence of polyspermy was high, ranging from 30 to 80% in the different groups studied. It is therefore concluded that the human oolemma provides little protection against multiple sperm fusion and that the frequency of gamete fusion is unexpectedly high, even when the spermatozoa are derived from infertile men.