A randomized controlled study of human Day 3 embryo cryopreservation by slow freezing or vitrification: vitrification is associated with higher survival, metabolism and blastocyst formation.

BACKGROUND: The aim of this study was to compare two methods of cryopreservation for the cleavage-stage human embryo: slow freezing and vitrification. METHODS: A total of 466 Day 3 embryos, donated with consent, underwent cryopreservation by either slow freezing in straws or vitrification using the cryoloop. The vitrification procedure did not include dimethyl sulfoxide, but rather employed ethylene glycol and 1,2-propanediol as the cryoprotectants. Survival, embryonic metabolism and subsequent development to the blastocyst were used to determine the efficacy of the two procedures. RESULTS: Significantly, more embryos survived the vitrification procedure (222/234, 94.8%) than slow freezing (206/232, 88.7%; P < 0.05). Consistent with this observation, pyruvate uptake was significantly greater in the vitrification group, reflecting a higher metabolic rate. Development to the blastocyst was also higher following vitrification (134/222, 60.3%) than following freezing (106/206, 49.5%; P < 0.05). In a separate cohort of 73 patients who had their supernumerary embryos cyropreserved with vitrification, the resulting implantation rate and clinical pregnancy rate were 30 and 49%, respectively. CONCLUSIONS: Analysis of metabolism revealed that vitrification had less impact on the metabolic rate of the embryo than freezing, which was reflected in higher survival rate and subsequent development in vitro. Excellent pregnancy outcomes followed the warming and transfer of vitrified cleavage-stage embryos. These data provide further evidence that vitrification imparts less trauma to cells and is, therefore, a more effective means of cryopreserving the human embryo than conventional slow freezing. Clinicaltrials.gov identifier: NCT00608010.

Severe cytoplasmic abnormalities of the oocyte decrease cryosurvival and subsequent embryonic development of cryopreserved embryos.

BACKGROUND: Abnormalities of oocyte morphology affect embryo quality and viability. Whether morphological abnormalities of the oocyte influence cryosurvival and further development of derived embryos is not known. The aim of this study was to compare cryosurvival and progression to the blastocyst stage of frozen-thawed embryos derived from normal and abnormal oocytes. METHODS: A total of 5292 Grade 1 and 2 embryos from 964 women were frozen, thawed and subsequently cultured up to the blastocyst stage. The study was performed on excess embryos from patients who did not opt for cryopreservation. Cryosurvival, progression to the blastocyst stage and hatching were correlated with morphological characteristics of the oocytes that embryos were derived from. RESULTS: Presence of a cytoplasmic abnormality of the oocyte significantly decreased cryosurvival. This detrimental effect was more pronounced in embryos derived from oocytes with vacuolar cytoplasm or with central granulation. Furthermore, these embryos did not have the potential to develop into good quality blastocysts or reach the hatching stage. On the other hand, presence of a single extracytoplasmic abnormality of the oocyte did not affect cryosurvival and the potential to develop into good quality blastocysts. Grade 2 embryos derived from oocytes with irregular shape or a large perivitelline space had decreased cryosurvival. However when these embryos survived cryopreservation, their potential to develop good quality blastocysts or to reach hatching stage was unaffected. CONCLUSIONS: Embryos derived from oocytes with vacuolar cytoplasm or central granulation do not seem to bear the potential to develop good quality blastocysts or to reach hatching stage after cryopreservation. The presence of extracytoplasmic abnormalities alone does not affect blastocyst development despite decreasing cryosurvival.

The effect of pronuclear morphology on embryo quality parameters and blastocyst transfer outcome.

BACKGROUND: Embryo quality may be accurately assessed as early as the pronuclear zygote phase, as shown in recent studies. However, it is not known whether good quality zygotes are destined to become good quality cleavage stage embryos and blastocysts. METHODS: In this retrospective study, 86 intracytoplasmic sperm injection-embryo transfer cycles were studied where each available embryo was scored from the zygote until the blastocyst stage. Embryonic normality parameters such as pronuclear pattern, early cleavage, cleavage stage embryo grade, the presence of embryos with > or =8 cells on day 3 and blastocyst quality were recorded. Embryo transfer was undertaken at the blastocyst stage and the outcome was studied according to the pronuclear pattern exhibited by the zygotes. RESULTS: Embryos that showed an ideal pronuclear pattern (0 PN pattern) cleaved earlier and faster and resulted in better quality cleavage stage embryos and blastocysts. The incidence of blastocyst formation was 72% in zygotes showing a 0 PN pattern, compared with 12.7% in zygotes with double pronuclear abnormality. Higher implantation and pregnancy rates were obtained when at least one blastocyst derived from a 0 PN pattern zygote was included in the set of embryos to be transferred. CONCLUSIONS: Our results indicate that the pronuclear pattern of the zygote is closely related to blastocyst formation and quality. Blastocysts derived from 0 PN zygotes have a higher potential for implantation.

Blastocyst-stage transfer of poor-quality cleavage-stage embryos results in higher implantation rates.

OBJECTIVE: To determine the feasibility and success of blastocyst-stage embryo transfers in patients having only fair and poor quality cleavage-stage embryos on day 3. DESIGN: Prospective case study with historic controls. SETTING: Tertiary care private hospital IVF center. PATIENT(S): A total of 158 day 5 embryo transfer cycles in patients with grade 3 and grade 4 cleavage-stage embryos. Control group consisted of 162 day 3 transfer cycles performed with embryos of similar quality. INTERVENTION(S): In vitro culture of embryos up to the blastocyst stage. MAIN OUTCOME MEASURE(S): The percentage of cycles that culminated in the transfer of at least one blastocyst and implantation and pregnancy rate related to the day of transfer. RESULT(S): In the day 3 transfer group, a mean of 5.2 embryos were replaced per patient. This was significantly more than the mean of 2.4 embryos that could be replaced on day 5 (P <.001). The clinical pregnancy rate per embryo transfer was 27.2% and 33.5% in the two groups, respectively (P >.05). The implantation rate per embryo was significantly higher in the day 5 transfer group (15% vs. 5.9%). The multiple pregnancy and abortion rates were similar between the groups. CONCLUSION(S): Transfer of fair and poor quality embryos at the blastocyst stage is feasible and is associated with higher implantation rates as compared to transfer of similar quality embryos on day 3.

Blastocyst transfer following intracytoplasmic injection of ejaculated, epididymal or testicular spermatozoa.

Recent studies indicate a strong paternal influence on embryo development and progression of the embryo to the blastocyst stage. The aim of this study was to compare, during extended culture, the in-vitro development of embryos resulting from intracytoplasmic sperm injection (ICSI) of ejaculated spermatozoa (group 1, n = 347), epididymal (group 2, n = 22) or testicular (group 3, n = 18) spermatozoa from obstructive azoospermic and testicular spermatozoa from non-obstructive azoospermic (group 4, n = 31) subjects. Fertilization and blastocyst formation rates were significantly lower in group 4 (P < 0.05). The incidence of expanded and hatching blastocysts was significantly lower in group 4 (P < 0.05). Overall in 93.2% ejaculate ICSI cycles, blastocysts were transferred on day 5. This was significantly higher than the 62% day 5 transfers in the non-obstructive azoospermic group (P < 0.05). Implantation rate per embryo was significantly higher in the ejaculate ICSI group compared with the other groups (P < 0.05). Clinical pregnancy per transfer was similar between groups; however, significantly fewer multiple pregnancies were encountered in the non-obstructive azoospermic group (P < 0.01). In conclusion, the source of the spermatozoa, most likely to be indicative of the severity of spermatogenic disorder, affects the rate of blastocyst formation and blastocyst implantation. Spermatozoa from non-obstructive azoospermic subjects, when utilized for ICSI, result in embryos that progress to the blastocyst stage at a lower and slower rate and implant less efficiently.

In-vitro spermatogenesis resumption in men with maturation arrest: relationship with in-vivo blocking stage and serum FSH.

We have shown previously that germ cells recovered from some men with maturation arrest can resume spermatogenesis in vitro and give rise to late elongated spermatids. This study relates the ability of germ cells to differentiate in vitro to the stage at which spermatogenesis is blocked in vivo and to the patient's serum FSH concentration. The presence of germ cells at different stages of spermatogenesis was assessed, before and after culture, by classical cytology, by fluorescence in-situ hybridization and by immunocytochemistry with a germline-specific marker. The proportion of cases of maturation arrest at the primary spermatocyte, secondary spermatocyte and spermatid stage in which in-vitro resumption of meiosis was achieved was 24.3% (9/37), 100% (3/3) and 51.1% (23/45) respectively. Serum FSH concentrations were higher than normal in most cases. However, lower values were measured in patients in whom in-vitro spermatogenesis was achieved compared with those in whom no progression was detected. These data show that, under the conditions of this study, germ cells from men with very high serum FSH concentrations (>20 IU/l) are less likely to resume spermatogenesis in vitro than those coming from men with only moderate increase (10-20 IU/l).

Progression to the blastocyst stage of embryos derived from testicular round spermatids.

Progression to the blastocyst stage of embryos derived from testicular round spermatids in men with non-obstructive azoospermia was studied. A total of 56 men were studied in whom partial spermatogenesis failure had occurred where only very few spermatozoa (fewer than the number of oocytes retrieved) were extracted from multiple testicular biopsy specimens. Oocytes remaining after intracytoplasmic injection of testicular spermatozoa (group 1) were injected with round spermatids (ROSI, group 2). Only embryos derived from group 1 were transferred. Remaining embryos were observed under culture for 8 days and their progression to the blastocyst stage was recorded. Of the 546 oocytes injected with testicular spermatozoa, 404 (73.9%) showed evidence of 2-pronuclear (2PN) fertilization. Injection of testicular round spermatids resulted in 2PN fertilization rate of 50% (P < 0.05). Using a four-point grading system, 53% of the good quality embryos (grade 1 or 2) in group 1 reached the blastocyst stage compared with 25% in group 2 (P < 0.05). The rate of progression to the blastocyst stage of grade 3 and grade 4 embryos was 46 and 8.5% in the two groups respectively (P < 0.05). Using a different three-point grading system for the blastocysts, 75.3% of the blastocysts in group 1 were either grade 1 or grade 2 and 24.7% were grade 3. However, in group 2 all blastocysts were grade 3. All embryos observed in group 1 reached the blastocyst stage by day 5 or 6 compared with 25% of the embryos reaching the blastocyst stage by this time in group 2. While 31.2% of the blastocysts in group 1 showed evidence of spontaneous hatching in vitro, none of the blastocysts in group 2 hatched. In conclusion, progression to the blastocyst stage occurred at a much lower and slower rate in embryos derived from testicular round spermatids. Furthermore, all blastocysts resulting from ROSI were of poor quality and none showed spontaneous hatching. These results may explain the dismal outcome associated with ROSI.

Outcome of testicular sperm retrieval procedures in non-obstructive azoospermia: percutaneous aspiration versus open biopsy.

The aim of this study was to evaluate whether the extraction of testicular spermatozoa with percutaneous versus open biopsy has an effect on the treatment outcome with intracytoplasmic sperm injection (ICSI) in men with non-obstructive azoospermia. Regardless of testicular size, follicle stimulating hormone concentration, and previous biopsy result, percutaneous testicular sperm aspiration (PTSA) using a 21-gauge butterfly needle was attempted first and if this failed testicular sperm extraction (TESE) was performed. In 63 men spermatozoa were found with PTSA whereas in 228 men TESE had to be undertaken. More men in the PTSA group had previously been diagnosed with hypospermatogenesis (82 versus 50%). Compared with the PTSA group, more men in the TESE group had germ cell aplasia (27 versus 10%) or maturation arrest (22 versus 8%). There was no difference between the groups regarding mean age of men and their partners, duration of stimulation, oestradiol concentration on the day of human chorionic gonadotrophin, number of oocytes retrieved, fertilization rate, and embryo quality between the two groups. The number of embryos transferred (4.38 versus 3.90) was significantly higher in the PTSA group (P < 0.05), reflecting the increased number of embryos available for transfer. Implantation rate per embryo was 20.7% in the PTSA and 13.3% in the TESE group (P < 0.05). Clinical pregnancy rates were 46 and 29% in the PTSA and TESE groups respectively (P < 0.05). Clinical abortion rates were similar (21.2 versus 24%). It is concluded that in men with non-obstructive azoospermia, easier sperm retrieval, which is most likely indicative of a more favourable histopathology, is associated with higher implantation rates per embryo.

Comparing two embryo transfer catheters. Use of a trial transfer to determine the catheter applied.

OBJECTIVE: To analyze the performance of two different embryo transfer catheters (Wallace and Frydman) in an in vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) program. STUDY DESIGN: Four hundred twenty-eight IVF or ICSI embryo transfer cycles were analyzed. A trial transfer was performed before the initiation of controlled ovarian hyperstimulation to determine the choice of embryo transfer catheter, Wallace or Frydman. Actual transfer was undertaken with the catheter chosen from the trial transfer. RESULTS: During actual embryo transfer, 214 (93.5%) of the intended 229 Wallace transfers were successful, and in 15 transfers the Frydman catheter was used. Of the intended 199 Frydman transfers, all were successful. Clinical pregnancy rate, implantation rate per embryo and ectopic pregnancy rate per transfer for the Wallace catheter were 41.6%, 16% and 0.9%, respectively. Respective rates for the Frydman catheter were 36.0%, 14.4% and 0.9% (P > .05 for all variables). Trial catheterization prevented most of the unanticipated procedural difficulties during the actual transfer. CONCLUSION: Both Wallace and Frydman catheters performed similarly, although there was a slight but nonsignificant increase in clinical pregnancy rates with the Wallace catheter.

Low-dose aspirin does not increase implantation rates in patients undergoing intracytoplasmic sperm injection: a prospective randomized study.

PURPOSE: The aim was to evaluate the effect of aspirin on pregnancy and implantation rates in an unselected group of patients undergoing intracytoplasmic sperm injection (ICSI). METHODS: Two hundred and seventy-nine patients were randomized to receive 80 mg of aspirin (n = 139) or no treatment (r = 136) starting from the first day of controlled ovarian hyperstimulation. RESULTS: Duration of stimulation, gonadotropin consumption, peak estradiol, number of oocytes retrieved, fertilization rate, cleavage rate, and number of embryos transferred were similar in the two groups. Implantation and clinical pregnancy rates were 15.6% and 39.6% versus 15.1% and 43.4% in aspirin treated and untreated groups, respectively (P > 0.05). CONCLUSIONS: Low-dose aspirin administration does not improve implantation and pregnancy rates in an unselected group of patients undergoing ICSI.

Elevated serum progesterone level on the day of human chorionic gonadotropin administration does not adversely affect implantation rates after intracytoplasmic sperm injection and embryo transfer.

OBJECTIVE: To evaluate the association between serum P levels on the day of hCG administration and the outcome of intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective case study. SETTING: Assisted reproduction unit of a tertiary care private hospital. PATIENT(S): Nine hundred eleven ICSI cycles that proceeded to ET were studied. INTERVENTION(S): The decision to administer hCG was based on serum E2 levels and follicle size. Serum P was measured from frozen sera obtained on the day of hCG administration. Cycles were stratified according to serum P levels of <0.9 ng/mL (n = 298) or > or =0.9 ng/mL (n = 613). This cutoff level was selected because it yielded the highest sensitivity and specificity according to a receiver operator characteristic curve. MAIN OUTCOME MEASURE(S): Implantation and clinical pregnancy rates. RESULT(S): In cycles with high serum P levels, more oocytes were retrieved and more embryos were available for transfer. Clinical pregnancy rates per ET in the low and high P groups were 36.9% and 45.4%, respectively (P<.05). The implantation rate per embryo was similar in the two groups (14.9% and 16.4%, respectively, in cycles with P levels <0.9 vs > or =0.9 ng/mL). Abortion rates were 22.7 and 25.8%, respectively (P>.05). CONCLUSION(S): Our data showed no adverse effect of high serum P levels on the day of hCG administration on implantation rates after ICSI and ET.