Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis.

BACKGROUND: Human mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification. METHODS: We used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation. RESULTS: Control, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90-100 µm in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3-4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8-9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied. CONCLUSIONS: Even though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane "recycling" that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardening.

Oocyte slow freezing using a 0.2-0.3 M sucrose concentration protocol: is it really the time to trash the cryopreservation machine?

OBJECTIVE: To update results on outcomes with frozen/thawed oocytes using a differential sucrose concentration during dehydration (0.2 M) and rehydration (0.3 M), combined with a one-step propanediol exposure. DESIGN: Retrospective cohort study. SETTING: Private IVF centers. PATIENT(S): Infertile couples undergoing IVF treatment. INTERVENTION(S): Oocyte thawing cycles between May 2004 and December 2010. MAIN OUTCOME MEASURE(S): Survival, fertilization, and cleavage rates were reported to evaluate biological outcomes. Clinical pregnancy and implantation rates were analyzed as markers of efficiency. RESULT(S): Three hundred forty-two patients and 443 cycles were monitored; the survival was 71.8%, fertilization 77.9%, and of the embryos obtained 83.8% were classified as grade 1 and 2. Three hundred ninety-four transfers were performed, resulting in 90 pregnancies. The pregnancy rate per transfer was 22.8% and per patient was 26.3%, with 122 gestational sacs. The implantation rate per embryo was 13.5%. Patients were divided into three groups according to their age: ≤ 34 years (group A), 35-38 years (group B), and ≥ 39 years (group C). Biological outcomes were comparable in all three groups, whereas the pregnancy rate per transfer was higher in the first group (27.7% vs. 21.4% and 17.6%). The implantation rates per injected egg were 11.8%, 8.0%, and 7.5% for the three groups, respectively. CONCLUSION(S): The biological and clinical data obtained on 443 cycles are consistent with our previous results showing that slow freezing of oocytes can be a valid tool in IVF practice when performed with a suitable protocol.

Fertility preservation in women after the cancer.

Thanks to the recent advances in cancer care, more and more young women can survive but suffer from infertility as a result of cancer treatment that had to be submitted. There are a variety of methods to preserve fertility, as chemoprotection, ovariopexy, and some assisted reproductive technologies, although some of these are promising but still highly experimental techniques. Cryopreservation of embryos for example is already established, while the oocyte banking is still considered an experimental practice. Many experiments have been conducted around the world on the cryopreservation of ovarian tissue and maturation of ovarian follicles, in an attempt to demonstrate its potential use in fertility preservation. Although in recent years there has been major improvements in the preservation of ovarian tissue, there are still many unresolved technical issues related to these procedures. In this chapter we examine the recent evidence of the pathophysiology of chemotherapy / radiotherapy-induced gonadal toxicity, and recent data regarding the indications and results of the techniques used to preserve fertility in women with cancer.

Factors affecting thawed oocyte viability suggest a customized policy of embryo transfer.

OBJECTIVE: To identify factors that might affect the clinical outcome of oocyte slow freezing. DESIGN: Retrospective study. SETTING: Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy. PATIENT(S): Patients with spare metaphase II cryopreserved oocytes performing 371 thawing cycles. INTERVENTION(S): Oocytes were cryopreserved by slow freezing<40 hours after hCG administration (group A) and >or=40 hours after hCG administration (group B). Thawed oocytes were inseminated by intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Clinical pregnancy, implantation, abortion, and delivery rates. RESULT(S): Clinical pregnancy rate per thawed cycle (PR) and implantation rate (IR) were significantly higher in group A compared with group B both in young (PR: 25% vs. 9.6%; IR: 18.9% vs. 8.8%) and in older patients (PR: 25% vs. 10.1%; IR: 17.5% vs. 6.7%). In the young patient subgroup, clinical pregnancy and implantation rates with three transferred embryos were higher in group A vs. group B (PR: 72.7% vs. 25%, and IR: 36.4% vs. 12.5%, respectively). This difference was not found in the subgroup of older patients. CONCLUSION(S): The timing at which oocyte cryopreservation is performed and the number of transferred embryos play a key role in the clinical outcome. The suggested cut-off time for cryopreservation is between 39 and 40 hours after hCG administration.

Impact of oocyte cryopreservation on embryo development.

OBJECTIVE: To verify whether the morphologic evaluation of zygotes and embryos derived from thawed oocytes could provide some relevant information regarding their developmental performance. DESIGN: Fertilization, zygote, and embryo morphology from sibling fresh and frozen oocytes was compared. SETTING: Reproductive Medicine Unit, Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. PATIENT(S): Two hundred thirty-four patients underwent intracytoplasmic sperm injection cycles from which 1,101 spare metaphase II oocytes were cryopreserved. Subsequently, 256 thawing cycles were performed, and 997 oocytes were thawed. INTERVENTION(S): Intracytoplasmic sperm injection was performed on both fresh and frozen oocytes. MAIN OUTCOME MEASURE(S): Fertilization rates, pronuclear zygote morphology, and embryo cleavage rates. RESULT(S): Thawed oocytes had lower chances of being fertilized and developing into top-quality zygotes and regularly cleaving embryos when compared with sibling fresh oocytes irrespective of female age. As a result, the percentage of transferred cycles was significantly lower in frozen cycles compared with fresh cycles (79% and 93%, respectively); the proportion of transferred top-quality embryos followed the same trend. CONCLUSION(S): Reduced fertilization and cleavage rates in frozen cycles when compared with sibling fresh oocytes suggest that, even if surviving thawing, the process of slow freezing has a negative impact on the potential of further growth that is evident as early as the first cleavage divisions.

Birefringence characteristics in sperm heads allow for the selection of reacted spermatozoa for intracytoplasmic sperm injection.

OBJECTIVE: To verify clinical outcome after injection of spermatozoa that have undergone the acrosome reaction (reacted spermatozoa) vs. those still having an intact acrosome (nonreacted spermatozoa). DESIGN: Prospective, randomized study. SETTING: Reproductive Medicine Unit, Italian Society for the Study of Reproductive Medicine, Bologna, Italy. PATIENT(S): According to a prospective randomization including 71 couples with severe male factor infertility, intracytoplasmic sperm injection (ICSI) was performed under polarized light that permitted analysis of the pattern of birefringence in the sperm head. Twenty-three patients had their oocytes injected with reacted spermatozoa, 26 patient's oocytes were injected with nonreacted spermatozoa, and in 22 patients both reacted and nonreacted spermatozoa were injected. INTERVENTION(S): Intracytoplasmic sperm injection was performed under polarized light to selectively inject acrosome-reacted and acrosome-nonreacted spermatozoa. MAIN OUTCOME MEASURE(S): Rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. RESULT(S): There was no effect on the fertilizing capacity and embryo development of either type of sperm, whereas the implantation rate was higher in oocytes injected with reacted spermatozoa (39.0%) vs. those injected with nonreacted spermatozoa (8.6%). The implantation rate was 24.4% in the group injected with both reacted and nonreacted spermatozoa. The delivery rate per cycle followed the same trend. CONCLUSION(S): Spermatozoa that have undergone the acrosome reaction seem to be more prone to supporting the development of viable ICSI embryos.

Embryo morphology and development are dependent on the chromosomal complement.

OBJECTIVE: To analyze embryo morphology in relation to the corresponding chromosomal status, in order to evaluate the effects of aneuploidy over fragmentation, delayed cleavage, and arrested cleavage. DESIGN: Retrospective study. SETTING: Reproductive Medicine Unit, Società Italiana di Studi di Medicina della Riproduzione, Bologna, Italy. PATIENT(S): A total of 662 patients with a poor prognosis for pregnancy underwent 916 cycles of preimplantation genetic diagnosis for aneuploidy. INTERVENTION(S): The chromosomes XY, 13, 15, 16, 18, 21, and 22 were analyzed in blastomeres biopsied from day 3 embryos. MAIN OUTCOME MEASURE(S): Embryo morphology, chromosomal status of the analyzed blastomeres, and spreading and reanalysis of nontransferred embryos. RESULT(S): The incidence of chromosomal abnormalities was significantly higher in arrested or slow-cleaving embryos, and in embryos cleaving too fast, compared to embryos with eight cells at 62 hours after insemination. The presence of an uneven number of blastomeres or fragments scattered in the perivitelline space was associated with an increased incidence of chromosomal abnormalities. CONCLUSION(S): A correlation between embryo development and chromosomal complement makes the incidence of chromosomal abnormalities significantly higher in embryos dividing according to a time frame and a symmetry plan which are different from what are expected. The type of fragmentation is also related to chromosomal status, which explains why the extrusion of fragments might severely affect embryo viability.

First meiosis errors in immature oocytes generated by stimulated cycles.

OBJECTIVE: To investigate chromosomal errors detected by first polar body (PB) biopsy in relation to the nuclear maturity of the oocytes. DESIGN: Retrospective study. SETTING: Reproductive medicine unit. PATIENT(S): Eighty-seven cycles were examined by PB biopsy for aneuploidy. Indications were maternal age >or=38 years (49 cycles), repeated IVF failures (22 cycles), and others (16 cycles). INTERVENTION(S): First polar bodies were analyzed for the chromosomes 13, 16, 18, 21, and 22 in both in vivo and in vitro matured oocytes. Euploid oocytes were inseminated by intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Chromosomal status of the analyzed oocytes, development after intracytoplasmic sperm injection, pregnancy, and implantation rates. RESULT(S): In in vitro matured oocytes, the proportion of chromosomal abnormalities was higher than in in vivo matured oocytes (70% vs. 54%, P<.005), with complex abnormalities being the prevailing defect (62% vs. 40%, P<.001). Conversely, the presence of an extra chromatid or the lack of a chromatid was more frequent in in vivo than in in vitro matured oocytes (55% vs. 34%, P<.001). CONCLUSION(S): The low viability of in vitro matured oocytes from stimulated cycles could be related to a significantly higher proportion of chromosomal abnormalities compared with in vivo matured oocytes. Complex abnormalities, involving two or more chromosomes, gave the strongest contribution to the detected increase.