Two hundred eighteen consenting patients entered a randomized study of the application of chemical zona pellucida thinning on their day 3 embryos, prior to uterine transfer. Of those control patients (n =108), whose embryos remained unmanipulated, 40 (37.0%) have ongoing/delivered pregnancies, while in the experimental group (n =110), whose embryos had their zonae pellucidae chemically thinned, there are 49 patients (44.6%) who have ongoing/delivered pregnancies. Although this difference is not significant, clearly the application of this micromanipulative intervention has not been detrimental, and this bodes well for routine application of embryonic micromanipulation procedures in general. Certain patient subgroups were studied including older women, those with elevated basal follicle stimulating hormone levels, patients with embryos of differing zona thickness, and patients with embryos of differing uniformity of zona thickness. No significant influence of chemical removal of the outside of the zona on the implantation rate of embryos in any of these subgroups was observed other than a marginally significant (P =0.095) improvement of implantation of embryos with less than 4.0 µm variation in zona thickness when chemical zona thinning was applied. Failure of chemical zona thinning to enhance human embryo implantation significantly, compared to assisted hatching by complete zona drilling, strongly suggests that the bilayered human zona pellucida needs to be fully breached, unlike that of the mouse.

The rate of successful implantation after replacement of frozen-thawed embryos in in vitro fertilization is commonly only 5% to 10% per embryo. A limiting factor may be inability of otherwise viable embryos to be released from the intact zonae pellucidae. Culture conditions and/or cryopreservation in in vitro fertilization may affect the zona and impair blastocyst hatching. Therefore opening of the zona by partial slicing by means of micromanipulation before replacement of early cleaved embryos may improve chances of eventual hatching (referred to as assisted hatching). In 65 thawed embryo replacement cycles methyl-prednisolone and antibiotics were given for 4 days mid cycle. Assisted hatching was performed in 32 cycles, with 33 cycles left as controls. Patient's age, infertility, cycle supplementation, and number of thawed and replaced embryos did not differ significantly between the two groups. Rates of viable embryonic implantation were 16% (10/63) and 9% (6/64) in the assisted hatching and control groups, respectively. Group sizes need approximately to double before this trend toward improved implantation with the use of assisted hatching reaches statistical significance.

The possibility of pronuclear gender determination using morphological criteria only was investigated in 140 two-pronucleate and 39 tripronucleate zygotes. Zygotes were videotaped on different focal points and the positions of the polar bodies, pronuclear diameters, number and distribution of nucleoli, and presence of sperm tail remnants were indicated on diagrams. The three known criteria used for recognition of the paternal pronucleus in rodent zygotes were investigated. These criteria are (a) the presence of sperm tail remnants, (b) an increased pronuclear diameter, and (c) the farthest distance from the second polar body. Sperm tail remnants were observed in only 3/342 (1%) of the pronuclei. Pronuclear diameters and positions of the largest pronuclei did not reveal any trends. Pronuclei of tripronucleate zygotes were frequently smaller than those of two-pronucleate ones. The parental origin of human pronuclei cannot be determined morphologically using standard light optics. Microsurgical removal of paternal pronuclei from polyspermic zygotes should therefore be implemented with caution.