Optimizing human oocyte cryopreservation for fertility preservation patients: should we mature then freeze or freeze then mature?

OBJECTIVE: To evaluate the maturation and post-thaw survival rates of immature oocytes to determine whether in vitro maturation (IVM) should be attempted prior to or after cryopreservation. DESIGN: Nonrandomized observational study. SETTING: Private academic and clinical reproductive center. PATIENT(S): Patients (n = 71) who donated immature unusable oocytes after vaginal oocyte retrieval (VOR) after undergoing controlled ovarian hyperstimulation using a standard GnRH antagonist protocol. INTERVENTION(S): Germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) oocytes (n = 175) were obtained from consenting IVF patients for fresh IVM, post-thaw IVM, or control group. In the fresh IVM group, GV- and MI- stage oocytes (n = 69) were cultured for 24 hours, matured in vitro (IVM-MII), cryopreserved, thawed, and evaluated for survival. In the post-thaw IVM group, GV- and MI- stage oocytes (n = 27) were frozen on day 0, thawed, evaluated for survival, and cultured for 24-hour IVM. MII donor oocytes (n = 79) were cryopreserved and thawed as a control. MAIN OUTCOME MEASURE(S): Survival postfreeze and oocyte development to the MII stage was analyzed using a χ(2) analysis. RESULT(S): Fresh IVM had a significantly higher maturation rate than post-thaw IVM. CONCLUSION(S): Oocyte cryopreservation is important for patients at risk of ovarian cancer, elective fertility preservation, and, potentially, for ovum donation. The superior maturation rate of GV and MI oocytes in the fresh versus post-thaw groups provides strong evidence for maturing oocytes to the MII stage before cryopreservation.

Nitrogen vapor shipment of vitrified oocytes: time for caution.

Oocyte cryopreservation for fertility preservation, in both medical and elective situations, has significantly increased as freezing technology has improved. Slow freezing techniques demonstrated ∼ 50-80% survival of mature oocytes, however vitrification with ∼ 97% survival has become the preferred method for oocyte cryopreservation around the world. Our work investigated the effect of transporting cryopreserved oocytes to and from a long-term storage facility. Our findings demonstrate that extra caution should be practiced for vitrified oocytes, especially when handling and transferring between shipping and long-term cryopreservation storage containers.

Commercially available enhanced in vitro maturation medium does not improve maturation of germinal vesicle and metaphase I oocytes in standard in vitro fertilization cases.

Improvements in in vitro maturation techniques have focused on culture optimization to increase oocyte maturation rates. Specialized culture media, now commercially available, did not perform significantly better than standard IVF culture media for maturation of immature oocytes in our normal IVF cases.

Identification and characterization of repopulating spermatogonial stem cells from the adult human testis.

BACKGROUND: This study was conducted to identify and characterize repopulating spermatogonial stem cells (SSCs) in the adult human testes. METHODS: Testes biopsies from obstructive azoospermic patients and normal segments of human testicular tissue were used. Flow cytometry, real-time PCR and immunohistochemical analysis were performed. Purified human spermatogonia were transplanted into busulfan-treated recipient mouse testes and integrated cells were detected by human nuclear protein antibody co-localized with stem cell and germ cell markers. RESULTS: Testicular biopsies collected from obstructive azoospermic men showed similar morphology and distribution of markers to the normal human testes. Flow cytometry showed distinct populations of stage-specific embryonic antigen-4 (SSEA-4), CD49f and CD90 positive cells in the adult human testes. SSEA-4 (+) cells showed high expression levels of SSC-specific genes and high levels of telomerase activity. Extensive colonization of human cells in the mouse testes indicates the presence of highly enriched populations of SSCs in the SSEA-4 (+) sorted cells. All the HNP (+) cells in the mouse testes were positive for germ cell marker dead box mRNA helicase and only half of them were dimly positive for c-kit. In addition, subpopulations of human spermatogonia that colonized mouse testes were positively stained for CD49f, GPR-125, Nanog and Oct-4 indicating the existence of population of cells among human spermatogonia with SSC and pluripotent characteristics. CONCLUSIONS: This study clearly demonstrates that repopulating human SSCs have phenotypic characteristics of SSEA-4(+), CD49f(+), GPR-125(+)and c-Kit (neg/low). The results have direct implications for enrichment of human spermatogonia for further culture and germ cell differentiation studies.

Monozygotic pregnancies conceived by in vitro fertilization: understanding their prognosis.

OBJECTIVE: To analyze the outcomes and particular characteristics of monozygotic (MZ) pregnancies conceived by in vitro fertilization (IVF). DESIGN: Retrospective data analysis. SETTING: Large private-academic fertility center. PATIENT(S): IVF-conceived MZ pregnancies. INTERVENTION(S): Statistical analysis of MZ pregnancy outcomes depending on fetal order and pregnancy reductions status. MAIN OUTCOME MEASURE(S): Spontaneous pregnancy reduction, pregnancy loss, take-home baby rate, perinatal mortality, gestational age at delivery, and birth weight. RESULT(S): A total of 72 of 3,426 pregnancies (2.1%) were MZ, and 70 were included in the study. Of these, 34 cases (48.5%) were high-order multiple pregnancies (HOMP), and 36 (51.5%) were non-HOMP. In the HOMP group, only 2.9% (1 of 34) had a complete pregnancy loss while 38.8% (14 of 36) of the non-HOMP were lost by 20 weeks' gestation. Of the HOMP patients, 73.1% therapeutically reduced the MZ component, and a statistically significant difference in gestational age of delivery (37.8 ± 3.2 vs. 28.1 ± 7.7) and birth weight (2796 ± 865.8 vs. 1110.0 ± 731.6) was seen when compared with nonreduced HOMP. CONCLUSION(S): Twinning with MZ is encountered in a small but important number of pregnancies derived from assisted reproduction. The prognosis for these patients is unfavorable, particularly for single-implantation MZ pregnancies and for nonreduced HOMP. Patients who do not spontaneously reduce a MZ-HOMP by 12 weeks may benefit from therapeutically reducing the MZ component of the pregnancy.

Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization.

A major challenge of assisted reproduction technologies (ARTs) is to mimic the natural environment required to sustain oocyte and embryo survival. Herein, we show that the ceramide-metabolizing enzyme, acid ceramidase (AC), is expressed in human cumulus cells and follicular fluid, essential components of this environment, and that the levels of this enzyme are positively correlated with the quality of human embryos formed in vitro. These observations led us to develop a new approach for oocyte and embryo culture that markedly improved the outcome of in vitro fertilization (IVF). The addition of recombinant AC (rAC) to human and mouse oocyte culture medium maintained their healthy morphology in vitro. Following fertilization, the number of mouse embryos formed in the presence of rAC also was improved (from approximately 40 to 88%), leading to approximately 5-fold more healthy births. To confirm these observations, immature bovine oocytes were matured in vitro and subjected to IVF in the presence of rAC. Significantly more high-grade blastocysts were formed, and the number of morphologically intact, hatched embryos was increased from approximately 24 to 70%. Overall, these data identify AC as an important component of the in vivo oocyte and embryo environment, and provide a novel technology for enhancing the outcome of assisted fertilization. Eliyahu, E., Shtraizent, N., Martinuzzi, K., Barritt, J., He, X., Wei, H., Chaubal, S., Copperman, A. B., Schuchman, E. H. Acid ceramidase improves the quality of oocytes and embryos and the outcome of in vitro fertilization.

Paternal age and assisted reproductive technology outcome in ovum recipients.

This study suggests that paternal age may be inversely associated with reproductive outcome, as demonstrated by a decline in fertilization, blastocyst formation, implantation and cryopreservation rates with advancing age.

Human blastocyst morphological quality is significantly improved in embryos classified as fast on day 3 (>or=10 cells), bringing into question current embryological dogma.

OBJECTIVE: To evaluate developmental potential of fast cleaving day 3 embryos. DESIGN: Retrospective analysis. SETTING: Academic reproductive center. PATIENT(S): Three thousand five hundred twenty-nine embryos. INTERVENTION(S): Day 3 embryos were classified according to cell number: slow cleaving: or=10 cells, and further evaluated on day 5. The preimplantation genetic diagnosis (PGD) results of 43 fast cleaving embryos were correlated to blastocyst formation. Clinical outcomes of transfers involving only fast cleaving embryos (n = 4) were evaluated. MAIN OUTCOME MEASURE(S): Blastocyst morphology correlated to day 3 blastomere number. Relationship between euploidy and blastocyst formation of fast cleaving embryos. Implantation, pregnancy (PR), and birth rates resulting from fast embryo transfers. RESULT(S): Blastocyst formation rate was significantly greater in the intermediate cleaving (72.7%) and fast cleaving (54.2%) groups when compared to the slow cleaving group (38%). Highest quality blastocysts were formed significantly more often in the fast cleaving group. Twenty fast cleaving embryos that underwent PGD, formed blastocysts, of which 45% (9/20) were diagnosed as euploid. Aneuploidy was diagnosed in 82.6% (19/23) of arrested embryos. A 50% implantation and 100% PR and birth rate were achieved with embryo transfers involving fast cleaving embryos. CONCLUSION(S): Fast cleaving embryos not only reach the blastocyst stage at a similar rate to intermediate cleaving embryos, but also exceed morphological quality criteria on day 5. Fast cleaving embryo transfers demonstrated a high clinical potential.

The morphology of extracted testicular sperm correlates with fertilization but not pregnancy rates.

OBJECTIVE: To investigate sperm morphology on the day of fresh testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI), and its effect on fertilization and pregnancy rates, as TESE in conjunction with ICSI results in high fertilization and pregnancy rates in most patients, but to our knowledge only one small study has assessed the morphology of retrieved sperm and found no correlation with the success of fertilization. PATIENTS AND METHODS: In a retrospective database analysis in a large academic centre, 68 men had 75 cycles of TESE combined with ICSI from January 2004 until April 2006. Sperm obtained by TESE was morphologically analysed at high (x 400-600) magnification and used for ICSI on the day of tissue retrieval. Sperm were classified as being either normal, having an amorphous head, having a mid-piece defect or having multiple defects. The calculated percentage of abnormal sperm injected was compared with the normal fertilization rate using Pearson's correlation coefficient, and pregnancy rates between groups were compared using chi-square analysis. RESULTS: Fifteen cycles had all morphologically normal sperm; 21 cycles had 50-99% normal forms and 39 cycles had <50% normal sperm. There was a highly significant correlation between the percentage of normal sperm used for ICSI and fertilization rates (P = 0.007). Overall, 43 clinical pregnancies resulted in this series, i.e. three among the group with all normal sperm injected, 12 in the group with 50-99% normal sperm and 28 in the group with <50% normal forms. There were also 11 pregnancies in cycles that used no normal forms. Pregnancy rates did not differ significantly among the groups (P = 0.08). CONCLUSIONS: TESE with ICSI frequently results in successful pregnancy; normal morphology was highly and significantly associated with successful fertilization, but importantly there were still 10 clinical pregnancies in cycles where only abnormal sperm were used. Sperm morphology after TESE should be assessed at the time of the procedure, and whenever possible, morphologically normal sperm chosen for injection. However, it is reassuring that acceptable fertilization and pregnancy rates are still achievable in cases with no morphologically normal sperm available.

Report of four donor-recipient oocyte cryopreservation cycles resulting in high pregnancy and implantation rates.

OBJECTIVE: To determine the clinical potential of donor-oocyte cryopreservation and thaw techniques for recipient patients. DESIGN: Institutional review board-approved prospective study of donor oocyte cryopreservation. SETTING: A large, private infertility center. PATIENT(S): Four anonymous oocyte donors underwent ovarian hyperstimulation for the purpose of oocyte retrieval and cryopreservation. The oocytes were subsequently thawed, fertilized, and transferred to 4 recipient patients. INTERVENTION(S): Oocytes were obtained from young donor patients and were cryopreserved with a slow freeze/rapid thaw protocol in which 1,2-propanediol (PrOH) and sucrose were used as cryoprotectants. Oocytes that survived were inseminated using intracytoplasmic sperm injection (ICSI). Resulting embryos were replaced into the recipient patients on the third day post-insemination. MAIN OUTCOME MEASURE(S): Post-thaw survival rate, fertilization rate, cleavage rate, implantation and clinical pregnancy rates. RESULT(S): A total of 79 metaphase II oocytes were frozen, stored frozen overnight in liquid nitrogen, and then thawed. The post-thaw survival rate was 86.1%. Normal fertilization following ICSI occurred in 89.7% of the surviving oocytes. Cleavage was observed in 91.8% of normally fertilized oocytes. A total of 23 embryos were transferred to 4 recipient patients. A clinical pregnancy rate of 75% and an implantation rate of 26.1% were achieved. CONCLUSION(S): Human oocyte cryopreservation is an effective technique that can be applied in clinical situations with high oocyte survival and clinical pregnancy rates expected.

Blastocyst embryo transfer is associated with a sex-ratio imbalance in favor of male offspring.

OBJECTIVE: To evaluate the sex ratio of offspring born after blastocyst transfers. DESIGN: Retrospective data analysis. SETTING: A large assisted reproductive technology center. PATIENT(S): We included 1,284 offspring from 937 deliveries during the period August 2003-August 2005. INTERVENTION(S): Tabulation and statistical analysis of all births resulting from fresh IVF cycles. The sex of resulting offspring was compared in both day 3 and blastocyst transfers for all births and for singleton deliveries. In addition, the sex of children conceived with the use of autologous oocytes and donor oocytes was evaluated. MAIN OUTCOME MEASURE: Sex ratio of offspring born following embryo transfers (ETs) after day 3 of culture and sequential blastocycst culture. RESULT(S): The overall sex ratio was significantly shifted toward males when blastocyst transfers were performed. Blastocyst transfers with only the use of autologous oocytes resulted again in a significantly higher proportion of male offspring. An even greater proportional difference was encountered in singleton offspring from donor oocytes. However, significance was not reached because of the limited number of offspring in the subgroup. CONCLUSION(S): This is the first individual-center report of a significant sex-ratio imbalance after the sequential media culture of blastocysts. The large imbalance in singleton births associated with the use of donor oocytes, although not significant, is cautionary in regard to the use of elective single ETs. Observation and publication of phenomena such as the effects of extended culture on the sex ratio of live-borns will allow us a better understanding of early differences in sexual dimorphism of the embryo, and will allow us to counsel our patients more appropriately.