Michael J. Tucker

Birth after cryopreservation of immature oocytes with subsequent in vitro maturation

OBJECTIVE To establish the clinical feasibility of using cryostored germinal vesicle oocytes for IVF and ET. DESIGN Case report. SETTING Private infertility clinic. PATIENT(S) A 28-year-old woman with tubal infertility undergoing IVF therapy. INTERVENTION(S) Oocytes collected after ovarian stimulation were frozen without insemination or were inseminated, fertilized, and frozen as cleavage stage embryos. No fresh oocyte or embryo transfer was undertaken. All oocytes were thawed, and those that survived were used for IVF-ET. MAIN OUTCOME MEASURE(S) Oocyte cryosurvival, in vitro maturation, fertilization, embryo development, and pregnancy outcome. RESULT(S) None of 16 mature oocytes survived thawing; however, three of 13 germinal vesicle oocytes survived. After 30 hours in vitro maturation two oocytes had matured and underwent intracytoplasmic sperm injection with the partners sperm. Both fertilized normally and were transferred to the patient. The woman delivered an apparently healthy female infant at 40 weeks. CONCLUSION(S) This case report proves the feasibility if not the efficiency of using immature oocytes for cryostorage, coupling both cryopreservation and in vitro maturation.

Potential Importance of Vitrification in Reproductive Medicine

Abstract As early as 1985, ice-free cryopreservation of mouse embryos at −196°C by vitrification was reported in an attempted alternative approach to cryostorage. Since then, vitrification techniques have entered more and more the mainstream of animal reproduction as an alternative cryopreservation method to traditional slow-cooling/rapid-thaw protocols. In addition, the last few years have seen a significant resurgence of interest in the potential benefits of vitrification protocols and techniques in human-assisted reproductive technologies. The radical strategy of vitrification results in the total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and in the surrounding solution (extracellular). The protocols for vitrification are very simple. They allow cells and tissue to be placed directly into the cryoprotectant and then plunged directly into liquid nitrogen. To date, however, vitrification as a cryopreservation method has had very little practical impact on human-assisted reproduction, and human preimplantation embryo vitrification is still considered to be largely experimental. Besides the inconsistent survival rates that have been reported, another problem is the wide variety of different carriers and vessels that have been used for vitrification. Second, many different vitrification solutions have been formulated, which has not helped to focus efforts on perfecting a single approach. On the other hand, the reports of successfully completed pregnancies following vitrification at all preimplantation stages is encouraging for further research and clinical implementation. Clearly, however, attention needs to be paid to the inconsistent survival rates following vitrification.

Day 3 morphology is a poor predictor of blastocyst quality in extended culture

OBJECTIVE To determine how the quality of blastocysts formed on day 5/6 of extended culture compares with their morphology on day 3. DESIGN Retrospective observational study of IVF laboratory records. SETTING Private assisted reproduction clinic. PATIENT(S) 101 IVF cycles in which 5 to 25 embryos were produced. The average maternal age was 33.1 years. INTERVENTION(S) Embryos were individually cultured in vitro in sequential media for an extended time to enable use of blastocysts for fresh transfer or cryopreservation. MAIN OUTCOME MEASURE(S) Comparison of embryo quality for putative ET or cryopreservation on day 3 with quality of embryos used for actual ET and cryopreservation on day 5/6. RESULT(S) Of 1,263 cleaving embryos, 559 were judged to have been suitable for use on day 3; 355 would have been used for ET (average per ET, 3.5) and 204 would have been frozen (equivalent to 44% utilization). In actuality, 471 blastocysts were used on day 5/6, of which 234 were transferred (average per ET, 2.3), and 237 were frozen (equivalent to 37% utilization). Only 48% embryos that would have been chosen for ET and/or cryopreservation on day 3 were eventually used in such a manner at the blastocyst stage. Historically, the rate of viable pregnancy from day 3 transfers was 30.5% per transfer; this rate increased to 45% with routine day 5/6 transfers. CONCLUSION(S) Extended culture of human embryos seems to increase discrimination of potential embryonic viability. Criteria for embryo selection on day 3 seem to be inadequate. Extended in vitro culture may therefore be an effective means of optimizing IVF clinical success.

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.

Recent developments in human oocyte, embryo and blastocyst vitrification: where are we now?

The target of any cryopreservation procedure should be to ensure high survival rates of living cells after thawing. Two important parameters determine the success of any cryopreservation protocol: the manner in which cells regain equilibrium in response to cooling, and the speed of freezing (cooling rate). Slow-rate freezing protocols result in the formation of ice crystals during cooling and warming. Vitrification, in which high cooling rates in combination with a high concentration of cryoprotectant are used, does not produce any ice crystals during cooling and warming. However, there is a practical limit to the attainable cooling speed, and also a biological limit to the concentration of cryoprotectant tolerated by the cells during vitrification. Although post-warming survival depends on the species, the developmental stage and the quality of the embryos being vitrified, it seems clear that vitrification methods are increasingly successful and might be a better method than slow cooling procedures in the field of cryobiology. Many of the potential problems and benefits underlying vitrification as a method of choice for embryo cryopreservation in clinical embryology will be discussed in this review.

Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette

The purpose of this study was to demonstrate the safety and efficacy of vitrification of human pronuclear stage (PN) embryos in the human assisted reproduction laboratory. Using single pronucleate (1PN) and three pronucleate (3PN) zygotes, the impact of vitrification in the Flexipet denuding pipette (FDP) as a carrier was assessed in terms of survival, embryonic development and blastocyst formation when compared according to the PN number, and unvitrified controls. A total of 65 1PN and 152 3PN zygotes were vitrified; after warming 82% (53/65) of 1PN and 90% (137/152) of 3PN survived. The overall percentage of warmed zygotes (1PN and 3PN) that cleaved and reached 2-cell stage did not differ (chi(2); P = 0.32) from the control groups (77%; 147/190 versus 85%; 115/136). In addition, when the cleavage behaviour was examined on day 3 for >or=4-cell stage, no significant differences (chi(2); P = 0.95) were observed between the vitrified group and the unvitrified control groups (74%; 109/147 versus 77%; 89/115). Comparing the developmental potential up to cavitation and blastocyst formation on day 5, the overall outcome of the vitrified PN was 31% compared with 33% for the controls (chi(2); P = 0.76). The simple vitrification protocol used in this study, and these data highlight the usefulness of vitrification using FDP as a consistent and effective cryopreservation method for pronuclear zygotes, and a suitable alternative to slow cryopreservation protocols.

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.

Trophectoderm grade predicts outcomes of single-blastocyst transfers

OBJECTIVE To estimate the effect of the embryo stage, trophectoderm (TE) morphology grade, and inner cell mass (ICM) morphology grade on live birth in single-blastocyst transfers. DESIGN Retrospective cohort study. SETTING Large private assisted reproductive technologies (ART) practice. PATIENT(S) Fresh autologous ART cycles. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Live birth. RESULT(S) A total of 694 single-blastocyst transfers met the inclusion criteria. Univariate regression analysis showed embryo stage and TE score to be correlated with implantation and live birth. Live birth rates were 57%, 40%, and 25% for TE grades A, B, and C, respectively. There was no significant association between ICM grade and implantation or live birth. Live birth rates were 53%, 52%, and 0% for ICM grades A, B, and C respectively. Multiple logistic regression analysis showed that only patient age and TE grade were significantly associated with implantation and live birth, whereas ICM grade was not significantly associated with outcome. The TE score had the strongest correlation with live birth. CONCLUSION(S) TE grading, but not ICM grading, significantly correlated with implantation and live birth for single-blastocyst transfers.

Successful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval

OBJECTIVE To evaluate a single treatment centers experience with autologous IVF using vitrified and warmed oocytes, including fertilization, embryonic development, pregnancy, and birth outcomes, and to estimate the likelihood of live birth of at least one, two, or three children according to the number of mature oocytes cryopreserved by elective fertility preservation patients. DESIGN Retrospective cohort study. SETTING Private practice clinic. PATIENT(S) Women undergoing autologous IVF treatment using vitrified and warmed oocytes. Indications for oocyte vitrification included elective fertility preservation, desire to limit the number of oocytes inseminated and embryos created, and lack of available sperm on the day of oocyte retrieval. INTERVENTION(S) Oocyte vitrification, warming, and subsequent IVF treatment. MAIN OUTCOME MEASURE(S) Post-warming survival, fertilization, implantation, clinical pregnancy, and live birth rates. RESULT(S) A total of 1,283 vitrified oocytes were warmed for 128 autologous IVF treatment cycles. Postthaw survival, fertilization, implantation, and birth rates were all comparable for the different oocyte cryopreservation indications; fertilization rates were also comparable to fresh autologous intracytoplasmic sperm injection cycles (70% vs. 72%). Implantation rates per embryo transferred (43% vs. 35%) and clinical pregnancy rates per transfer (57% vs. 44%) were significantly higher with vitrified-warmed compared with fresh oocytes. However, there was no statistically significant difference in live birth/ongoing pregnancy (39% vs. 35%). The overall vitrified-warmed oocyte to live born child efficiency was 6.4%. CONCLUSION(S) Treatment outcomes using autologous oocyte vitrification and warming are as good as cycles using fresh oocytes. These results are especially reassuring for infertile patients who must cryopreserve oocytes owing to unavailability of sperm or who wish to limit the number of oocytes inseminated. Age-associated estimates of oocyte to live-born child efficiencies are particularly useful in providing more explicit expectations regarding potential births for elective oocyte cryopreservation.

Chromatin fluorescence characteristics and standard semen analysis parameters: correlations observed in andrology testing among 136 males referred for infertility evaluation

This paper aims to describe the relation between standard semen analysis parameters (concentration, motility and morphology) and sperm chromatin structure assay (SCSA) results among patients referred for infertility evaluation. Healthy males (n = 136) seeking infertility consultation were evaluated prospectively by semen analysis and sperm chromatin structure assay (SCSA). Significant inverse correlations were observed between high sperm concentration and DNA fragmentation index (DFI) and high DNA stainability (HDS) (r =− 0.45; P < 0.001, and r =− 0.40; P < 0.001, respectively). Both progressive motility and normal morphology were also strongly inversely correlated with DFI and HDS. However, in stratified analysis the correlation between concentration ⩽ 20 M/ml and DFI, and concentration ⩽ 20 M/ml and HDS were not significant (P = 0.31 and 0.38, respectively). For men with sperm motility ⩽ 40% the correlation between motility and HDS was not significant (P = 0.22), but between motility and DFI the correlation remained significant (P = 0.04). Although strong correlations between DFI, HDS and semen analysis findings were noted in the overall study population, when oligozoospermic and asthenozoospermic patients were analysed separately the correlation between concentration and sperm chromatin fragmentation was not significant. For such men, SCSA appears to be a diagnostic variable independent of the semen analysis, providing information about nuclear abnormalities not readily apparent from standard semen analysis alone. Additionally, SCSA data may offer explanations for previous miscarriage, providing closure for some couples contemplating future use of anonymous donor sperm.