M.C. Schiewe

Enzymatic characterization of zona pellucida hardening in human eggs and embryos

PurposeTo characterize possible hardening of the human zona pellucida (ZP) and evaluate the effect of culture duration, patient age, and ZP thickness, ZP of unfertilized eggs (experiment 1, n =367; experiment 2, n =174) and abnormal embryos (experiment 1, n=52) were randomly designated for α-chymotrypsin treatment after 0, 24, 48, 72, 96, 120 h (experiment 1) and 48 h, 72 h, and 1 week (experiment 2) of in vitroculture in HTF medium supplemented with 0.5% human serum albumin. Mean ZP thickness was predetermined in experiment 2.MethodsThe dispersion of the ZP glycoproteins was assessed, and the duration of time for complete digestion was recorded as an index of ZP hardness.ResultsIn experiment 1, enzyme digestion duration increased (P<0.05) in the first 24 h in vitrofrom 18.0±2.0 to 34.6±2.5 min, and tended to decrease over the next 4 days in culture (25.2±1.3, 29.4±0.9, 27.3±0.6, 26.6+1.1, and 20.7±1.5 min on Day 2– 6 ZP, respectively). Zona hardening of fertilized eggs was revealed by a longer (P<0.01) digestion time (32.2±1.8 vs 25.8±0.6 min).ConclusionsThere were significant patient-to-patient variations (16.4±0.7 to 39.6±2.2 min); however, age was not correlated to enzyme digestion duration. In experiment 2 we determined that ZP thickness (range 8.4–21.6 μm; mean 14.6±0.2 μm) was not correlated (r=0.09) to the digestion interval (mean 24.3 ± 0.8 min). Based on our enzymatic ZP digestion measurements, it is apparent that spontaneous zona hardening does occur within 24 h of in vitroculture, similar to levels achieved postfertilization. The data do not support, however, the concept that additional, abnormal hardening of the ZP occurs during extended culturing.

Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system

PurposeA noncontact holmium:yttrium scandium gallium garnet (Ho:YSGG) laser system has been designed and tested for the micromanipulation of mammalian embryos. The purpose of this preliminary investigation was to determine the effectiveness of this laser for assisted hatching and evaluate its impact on embryo viability. The Ho:YSGG system, utilizing 250-μsec pulses at a wave-length of 2.1 μm and 4 Hz, was used to remove a portion of the zona pellucida (ZP) of two-to four-cell FVB mouse embryos.ResultsIn the first experiment there was no difference in blastocyst production or hatching rates following laser or conventional assisted hatching (LAH or AH, respectively) in contrast to control embryos cultured in a 5% CO2 humidified air incubator at 37°C. In the second experiment a blastocyst antihatching culture model was employed and LAH-treated embryos were cultured in a serum-free HTF medium (HTF-o). Blastocyst formation was not influenced by LAH treatment and hatching was increased (P < 0.01) from 4 to 60% compared to HTF-o control group.ConclusionsThese preliminary data demonstrate the utility and nontoxic properties of the Ho:YSGG laser system for quick and precise ZP drilling.

Potential risk of monochorionic dizygotic twin blastocyst formation associated with early laser zona dissection of group cultured embryos

OBJECTIVE To document the risk of in vitro monochorionic dizygotic twin formation in the implementation of a program of blastocyst biopsy with preimplantation genetic screening (PGS). DESIGN Case report. SETTING Private infertility laboratory. PATIENT(S) Prospective PGS patients with intracytoplasmic sperm injection-derived, group-cultured blastocysts over a 3-year period. INTERVENTION(S) Group culture in Global medium (Life Global) to optimize blastocyst formation of zygotes produced for blastocyst biopsy for PGS (n ≤ 8 embryos/25 μL droplet), and laser zona dissection (LZD) of all day-3 cleaved embryos to promote pre-expansion trophectodermal extrusion at the blastocyst stage (i.e., premature hatching). MAIN OUTCOME MEASURE(S) Blastocyst formation and quality grading on days 5 and 6 of in vitro culture for the vitrified embryo transfer of single or dual euploid blastocysts. RESULT(S) Over 3,000 blastocysts were produced in vitro. On two separate occasions, complete trophectodermal amalgamation was observed between two hatching blastocysts. Vitrified single-euploid blastocyst transfers efficiently implanted and established clinical pregnancies similar to dual-euploid blastocyst transfers, without the risk of twin formation. CONCLUSION(S) The amazing occurrence of monochorionic dizygotic twin formation has now been documented in vitro, supporting the theory that assisted reproductive technology may facilitate this rare perinatal condition. Furthermore, we have provided clinical evidence that the transfer of a single-euploid blastocyst can optimize a patients pregnancy success while reducing potentially undesirable conditions associated with monochorionic twin pregnancies.

Vitrification: the pioneering past to current trends and perspectives of cryopreserving human embryos, gametes and reproductive tissue

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Physiological characterization of blastocyst hatching mechanisms by use of a mouse antihatching model*†*Presented in part at the 42nd Annual Meeting of The Pacific Coast Fertility Society, Palm Springs, California, April 21 to 24, 1994.†Supported in part by a Department of Obstetrics and Gynecology Memorial Health Services grant at the University of California Irvine and by resources made available at the University of California Irvine-Beckman Laser Institute.

OBJECTIVES To use the protein-free medium blastocyst antihatching model to characterize physiological events that mediate hatching. DESIGN In a series of four prospective experiments, our aims were to [1] test the efficacy of the antihatching model and assisted hatching; [2] exam the influence of initial in vivo developmental stage and late serum supplementation on hatching inhibition; [3] discount the role of zona hardness and physical expansion directly affecting hatching; and [4] provide evidence that the trophectoderm is directly responsible for secreting a zona lysin. SETTING University-based research laboratory. RESULTS Culturing two- to eight-cell mouse embryos in serum-free human tubal fluid (HTF) medium significantly reduced hatching levels to < or = 2%, however, hatching increased to 10.7% when initially culturing morula-stage embryos. Hatching was effectively rescued to control levels when embryos were placed in HTF with serum at the early blastocyst stage. There was no difference in blastocyst total cell numbers or zona pellucida digestion intervals between culture treatments. Finally, we showed that trophectodermal vesicles, devoid of inner cell mass, are capable of hatching under control conditions. CONCLUSIONS The primary mechanism of blastocyst hatching is not physical expansion and abnormal zona hardness is not responsible for hatching inhibition. Certain extracellular precursors found in serum (e.g., amino acids) are required in culture medium upon cellular determination of trophectoderm (i.e., morula to blastocyst stage) to facilitate the intrinsic secretion of an undefined hatching factor.