Sharon R. Wiker

Partial dissection of the zona pellucida of frozen-thawed e human embryos may enhance blastocyst hatching, implantation, and pregnancy rates

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. Patients 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.

Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate

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.

Electroejaculation in combination with in vitro fertilization and gamete micromanipulation for treatment of anejaculatory male infertility

Objective: Failure to ejaculate may be overcome by use of electroejaculation. However, such semen samples are often unsuitable for therapies like intrauterine insemination. The combination of electroejaculation with in vitro fertilization, including gamete micromanipulation, should improve chances of fertilization and pregnancy in such cases. Study Design: Within a private infertility clinic electroejaculation in combination with intrauterine insemination was carried out in 18 cycles (10 couples). Four couples went on to receive therapy by electroejaculation plus in vitro fertilization, along with six other couples (15 cycles total) with semen too poor for intrauterine insemination. Results: One term pregnancy arose in the electroejaculation-intrauterine insemination group, and one term pregnancy plus one continuing pregnancy arose from two couples (three cycles) who underwent in vitro fertilization with conventional insemination after electroejaculation. Six couples (nine cycles) had embryos arising only from gamete micromanipulation transferred, and this yielded two term pregnancies, one spontaneous abortion, and a biochemical pregnancy. Two couples (three cycles) failed to achieve fertilization even with micromanipulation; however, donor-inseminated eggs gave rise to two term pregnancies and one continuing pregnancy in these patients. Conclusions: This report confirms the feasibility of in vitro fertilization in conjunction with electroejaculation and extends the therapy to incorporate gamete micromanipulation.

Duration of storage of cyropreserved human embryos

SummaryThe incidence of cell injury, embryo survival, and implantation following cryopreservation of zygotes and two- to five-cell embryos was studied in 100 patients in order to evaluate the effect of duration of storage. The incidence of individual cell survival was 58% regardless of the length of time kept in liquid nitrogen or the stage of the embryo at freezing. There were 104 of 208 (50%) thawed embryos that survived completely intact, and of those, 24 implanted successfully. Twenty-one (21%) patients had a clinical pregnancy; two of them miscarried. Neither the survival of zygotes or cleaved embryos upon thawing nor the incidence of implantation was affected by the duration of cryostorage.

Recognition of paternal pronuclei in human zygotes

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.

Treatment of male infertility and idiopathic failure to fertilize in vitro with under zona insemination and direct egg injection

Objective: Failure to fertilize eggs in vitro may be countered by micromanipulation of gametes to place selected spermatozoa underneath the zona pellucida of the egg or directly into the egg, thereby improving chances of fertilization and production of viable embryos. Analysis of our clinical data for assisted fertilization was undertaken to assess those factors of relevance in this therapy, and a description of our procedures are given. Study Design: Retrospective analysis of 85 cycles (73 couples) of in vitro fertilization and embryo transfer performed at a private infertility clinic, in which micromanipulation for assisted fertilization was used to overcome either severe male factor infertility or idiopathic failure to fertilize, was performed. Results: In 60 cycles where only embryos from under zona insemination were available for uterine transfer, 15 singleton and two twin pregnancies occurred (28.3% viable pregnancy rate per transfer, 14.1% embryonic implantation). In 14 of these cycles embryos arose only after repeated under zona insemination adding more spermatozoa; this accounted for four of the singleton and one of the twin pregnancies (38.5% pregnancy rate, 22.2% embryonic implantation). No embryos arose from partial zona dissection performed in five cycles on sibling eggs. However, in 16 cycles conventional insemination yielded fertilization in six cycles, and mixed transfer of these embryos and sibling embryos from under zona insemination gave rise to one pregnancy from four transfers (pregnancy rate 25%, embryonic implantation 7.1%). Likewise, in nine cycles donor spermatozoa yielded fertilization in eight cycles, and mixed transfer with sibling embryos fertilized by under zona insemination with partners spermatozoa gave rise to two pregnancies from five transfers (pregnancy rate 40%, embryonic implantation 15.8%). Fertilization and pregnancy rates did not differ whether couples suffered either from male factor infertility or from previous idiopathic fertilization failure. Direct egg injection of a single spermatozoon into 105 eggs gave an 88.6% egg survival and 32.3% fertilization. Mixed transfers with sibling embryos from conventional and under zona insemination yielded one triplet, one twin, and three singleton pregnancies. Conclusions: Overall, a 24.7% (21/85) viable pregnancy rate per cycle initiated occurred when only embryos from assisted fertilization were available. This strongly indicates that assisted fertilization made a real contribution in cases where either insufficient spermatozoa were available for conventional insemination or in cases where previous fertilization failure had arisen. The wide range of seminal parameters were found to be unhelpful in defining chances of success with assisted fertilization.

Coculture of Human Zygotes on Fetal Bovine Reproductive Tract Cells

When early cleaved human embryos are kept in culture, only one in four can be expected to develop into fully expanded blastocysts.1 Alternatively, only 1% to 12% of them will implant and develop into full-term babies, when replaced into the uterus or fallopian tube before the third cleavage division commences.2,3 Embryonic wastage following assisted reproduction can only in part be explained by an increased incidence of genetic abnormalities or loss at the time of replacement. Other more esoteric factors, like a reduced receptivity of the endometrium in stimulated menstrual cycles, probably play an important role as well.

Partial Zona Dissection for Enhancement of Sperm Passage through the Zona Pellucida

Fertilization in mammals occurs in a number of intricate steps, culminating in the union of male and female genomes. Spermatozoa capacitate prior to sperm receptor binding on the zona pellucida (ZP), where the acrosome reaction is induced. Following ZP penetration membrane fusion occurs, triggering oocyte activation. This leads to the release of cortical granules and the zona reaction, causing a slow, but usually permanent block to polyspermy. Changes in the oolemma may initiate a fast, but often weak and temporary block, as well. Following decondensation and syngamy, the fertilization process is completed with the formation of a genetically new conceptus at the two-cell stage.1 Pathological changes may occur during any of these steps, and fertilization may either be discontinued or result in a genetically abnormal embryo. Discontinued fertilization is common during human in vitro fertilization (IVF), especially when there is a sperm disorder. Although the methods for microsurgical fertilization proposed in recent years only alleviate abnormal ZP-binding, ZP-penetration, and/or membrane fusion, they may be welcome additions to IVF. Basically, three methods, each with its own advantages and disadvantages, have been proposed: