J. Goldfarb

Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis

Embryo cryopreservation is an important aspect of assisted reproduction. Many methods have been described, but they have been poorly investigated in randomized trials, highlighting the need for a systematic review of the literature. Meticulous electronic/hand searches were performed to locate randomized trials (RCT) comparing embryo cryopreservation methods. Primary outcomes were clinical pregnancy rate (CPR) and incidence of congenital abnormalities. Secondary outcomes included live-birth (LBR), ongoing pregnancy (OPR), implantation (IR), and miscarriage (MR) rates. Data were extracted to allow for an intention-to-treat analysis and analysed using a random-effects model. Literature search revealed 11 RCT, of which five were excluded. The quality of the included studies was variable, but generally poor. There was a significantly higher CPR, OPR and IR with vitrification compared with slow freezing (odds ratio (OR)=1.55, 95% confidence interval (CI)=1.03-2.32, OR=1.82, 95% CI=1.04-3.20 and OR=1.49, 95% CI=1.03-2.15, respectively). In addition, there was a significantly lower CPR and OPR with embryo ultra-rapid freezing compared with slow freezing (OR=0.35, 95% CI=0.16-0.76 and OR=0.37, 95% CI=0.17-0.81, respectively). Vitrification is superior to slow freezing, which in turn is superior to ultra-rapid freezing. However, more well-designed and powered studies are needed to further corroborate these findings.

Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births

Vitrification technology has shown great promise for cryopreservation of human embryos. The majority of this work has been with blastocyst-stage embryos. This report describes initial clinical results following vitrification of human embryos on day 3 of culture at the 6- to 8-cell stage. A total of 236 embryos were cryopreserved on cryoloops using a vitrification protocol. Warmed embryos were cultured until day 5 before transfer to the patient. The post-warming survival rate was 85%. The clinical pregnancy rate was 44% (34/77), and the implantation rate was 20% (40/201). In transfers where at least one warmed embryo reached the blastocyst stage by the day of transfer, the clinical pregnancy rate was 58% (28/48). The cryoloop was an excellent vessel for ultra-rapid cryopreservation of embryos. This study supports the effectiveness of a dimethylsulphoxide/ethylene glycol cryoprotectant combination for vitrification of human embryos at the 6- to 8-cell stage.

Does severe teratozoospermia affect blastocyst formation, live birth rate, and other clinical outcome parameters in ICSI cycles?

OBJECTIVEnTo determine if strict morphology correlates with outcome parameters in couples undergoing intracytoplasmic sperm injection (ICSI).nnnDESIGNnRetrospective review.nnnSETTINGnAcademic nonprofit IVF center.nnnPATIENT(S)nCouples undergoing IVF/ICSI.nnnINTERVENTION(S)nIn vitro fertilization and ICSI.nnnMAIN OUTCOME MEASURE(S)nSamples were evaluated for total sperm count, motlity, progression, and morphology using Krugers strict criteria. The ICSI cycle outcome parameters included fertilization, clinical pregnancy, implantation, live birth, and blastulation rates and blastocyst quality.nnnRESULT(S)nFertilization rates were high (74%-77%), and clinical pregnancy rates ranged from 60% (subgroup with 0% normal sperm) to 56% (subgroup with >/=7% normal forms). The highest pregnancy and live birth rates were observed in eggs fertilized with sperm from specimens with the most severe teratozoospermia. The blastulation rate was similar among subgroups. The percentage of high-quality blastocysts was significantly greater in the severely teratozoospermic patients compared with patients with >/=5% normal sperm (37% vs. 28%). This is likely because in the lower morphology subgroups, female factors are less prevalent and the primary infertility problem is male factor.nnnCONCLUSION(S)nThese data suggest that we reconsider the diagnostic value of strict morphology in assisted reproductive technology cycles involving ICSI. Sperm morphology assessed by Krugers strict criteria had little prognostic value in ICSI cycle outcomes. Sperm morphology did not appear to influence blastocyst development or blastocyst morphology. Microscopic selection of sperm with normal morphology during the ICSI procedure allowed excellent outcomes even in samples with severe teratozoospermia.

Secretion of soluble HLA-G by day 3 human embryos associated with higher pregnancy and implantation rates: assay of culture media using a new ELISA kit

Identification of chemical markers in human embryo culture media that relate to embryo quality and implantation potential could be an invaluable tool in embryo selection for transfer. Embryonic secretion of soluble human leukocyte antigen G (sHLA-G) has been postulated to be a marker for embryonic competence. This study examines sHLA-G concentrations in day 3 culture media droplets of embryos that were selected for transfer, as well as those being cryopreserved. A total of 712 embryo culture supernatants from 83 patients were assayed. Soluble HLA-G was detected in 306 of the 712 samples tested. In the 58 transfers in which at least one embryo selected for embryo transfer was positive for sHLA-G, the pregnancy rate was 64% (37/58) and the implantation rate per embryo transferred was 38%. In contrast, patients receiving only embryos that were negative for sHLA-G had both a lower pregnancy rate of 36% (9/25; P < 0.05) and a decreased implantation rate (19%; P < 0.05). Expression of sHLA-G was also related to increasing cell stage. Concentration of sHLA-G in embryo culture media was variable and in the low range (3-10 ng/ml). These data suggest an association between implantation potential and embryonic secretion of sHLA-G. The commercial assay kit utilized allowed for same day assessment of sHLA-G secretion. Addition of sHLA-G status to traditional morphological criteria may be useful as a clinical tool for embryo selection.

Single sperm cryopreservation on cryoloops: an alternative to hamster zona for freezing individual spermatozoa

Methodology enabling cryopreservation of individual spermatozoa in extreme cases of oligozoospermia would tremendously benefit patients. This study explores the use of a nylon loop for cryopreservation of small quantities of spermatozoa, and also describes a novel technique for freezing individual spermatozoa with this cryoloop. Experiments were conducted to compare sperm recovery and viability after cryopreservation in conventional vials versus on the cryoloops. Discarded human sperm specimens with varying parameters were utilized. The study also examines two different glycerolbased cryoprotectants, with and without test yolk buffer. For single sperm cryopreservation, 5-10 spermatozoa were selected and loaded onto cryoloops with the aid of a microscope and micromanipulation equipment. Sperm function testing was performed on both human and bovine spermatozoa frozen on cryoloops. Microquantities of spermatozoa frozen on cryoloops exhibited overall motility and viability parameters similar to control samples frozen in cryovials. Individually selected spermatozoa cryopreserved on loops were easily recovered and post-thaw motility was generally good. Sperm function testing demonstrated that both human and bovine spermatozoa cryopreserved on loops were able to undergo sperm head decondensation when injected into oocytes. Cryoloops may be an excellent alternative to hamster zonae for cryopreserving small numbers of human spermatozoa.

Granulocyte-macrophage colony stimulating factor (GM-CSF) and co-culture can affect post-thaw development and apoptosis in cryopreserved embryos

Purpose: The objective of this study was to evaluate the effects of growth factor supplementation and Vero cell co-culture on apoptosis and development of frozen thawed one-cell mouse embryos.Methods: The following treatment regimens were assessed: (a) control medium (b) Vero cell co-culture and (c) growth factor supplemented medium. The individual growth factors tested were: GM-CSF, IGF-I, IGF-II, TNF-α, FGF-4, LIF, TGF-α, TGF-β, IL-6, PDGF and EGF. Blastocyst development and differentiation were monitored. At termination of the experiments, overall blastomere number and apoptosis were assessed using the TUNEL assay.Results: No differences were observed in blastulation and hatching rates. ICM differentiation in thawed embryos was notably improved with either co-culture or growth factor supplementation. The only growth factor significantly modulating apoptosis in thawed embryos was granulocyte-macrophage colony stimulating factor (GM-CSF). GM-CSF enhanced continued cell survival and prevented apoptosis but did not influence overall cell number in developing blastocysts. Vero cell co-culture significantly increased cell number in blastocysts (124±42 vs 100±44 in control; P<0.05). Embryonic apoptosis was higher in the co-cultured embryos. The increased presence of apoptotic cells in blastocysts of high cell number may reflect the regulatory role of apoptosis in balancing ICM: TE ratios.Conclusion: These data indicate that culture conditions can modulate post-thaw embryonic development and apoptosis.

Examination of frozen cycles with replacement of a single thawed blastocyst

This investigation examined frozen cycle outcomes with the transfer of a single thawed blastocyst. It also aimed to determine if the suggested model could aid in achieving a better understanding of factors that influence success with frozen blastocyst transfer. A retrospective analysis was conducted of single frozen embryo transfer cycles carried out at the Cleveland Clinic Fertility Centre in Beachwood, OH. Between January 2001 and March 2004, 88 thaw procedures were initiated that resulted in 75 frozen cycles in which only a single thawed blastocyst was transferred. In 66 of these 88 thaw procedures, only a single embryo was available for thawing. The post-thaw survival rate of a single frozen blastocyst was 85% (56/66). These 56 frozen transfers with a single thawed blastocyst resulted in a clinical pregnancy rate per transfer of 27% (15/56) and a live birth rate of 18%. Blastocyst post-thaw morphology at transfer was found to be an important prognostic factor associated with success. The ability to re-expand within a few hours of the thaw appeared to be a strong indicator of blastocyst potential. Blastocyst age at cryopreservation did not impact outcomes. The single frozen embryo transfer model can yield valuable information and help gauge the effectiveness of a laboratorys cryopreservation protocol.

MOUSE OVARIAN FOLLICLE CRYOPRESERVATION USING VITRIFICATION OR SLOW PROGRAMMED COOLING: ASSESSMENT OF IN VITRO DEVELOPMENT, MATURATION, ULTRA-STRUCTURE AND MEIOTIC SPINDLE ORGANIZATION

Aim:u2002 To compare different outcomes of vitrification and slow freezing of isolated pre‐antral follicles and to evaluate different cryo‐devices for vitrification of isolated follicles.

Severe teratozoospermia and its influence on pronuclear morphology, embryonic cleavage and compaction

BackgroundFertilization, cell division and embryo development depend on genomic contributions from male and female gametes. We hypothesize that teratozoospermic sperm influences early embryo development and embryo compaction.MethodsWe conducted a retrospective analysis of embryos derived from intracytoplasmic sperm injection (ICSI) cycles. Two hundred thirty-five consecutive ICSI cycles were included in the study; all treatment was provided at the Cleveland Clinic Fertility Center. Patient cycles were divided by sperm morphology based on Krugers strict criteria: Group A, embryos where teratozoospermic sperm (0-2% normal) were used for ICSI and Group B, embryos where dysmorphic sperm (5-13% normal) were used for ICSI. All cycles analyzed were of patients doing day 3 embryo transfers. Outcome measures assessed included pronuclear (PN) pattern, syngamy, early cleavage, cell number, rate of compaction and blastulation of embryos left in culture and not transferred on day 3.ResultsA total of 1762 embryos were analyzed. PN patterns were similar in Group A and Group B embryos. No differences were noted in syngamy, cleavage, cell number or blastulation rate. Studying the development of embryos in culture after day 3 transfer revealed a difference in the timeline for compaction. By day 4, 25% of Group A embryos had compacted compared to 36% in Group B (P = 0.0007). There was no difference found between Group A and Group B embryos in regards to blastulation.ConclusionsWe did not find an association between sperm morphology and clinical outcomes. The impact of teratozoospermia may be masked in ICSI cycles where fertilization, implantation rate and clinical pregnancy rate are the primary outcome measures. However, by examining the timeline of development, we were better able to discern a potential paternal effect at critical transition points from fertilization through activation.

Paternal effect on genomic activation, clinical pregnancy and live birth rate after ICSI with cryopreserved epididymal versus testicular spermatozoa

BackgroundThis study takes an in depth look at embryonic development, implantation, pregnancy and live birth rates with frozen epididymal and testicular sperm from obstructed (OA) and non-obstructed (NOA) patients.MethodsPaternal effect of sperm source on zygote formation, embryonic cleavage, and genomic activation were examined. Additional outcome parameters monitored were clinical pregnancy rate (CPR), implantation rate (IR) and live birth rate.ResultsIn this report, we retrospectively analyzed 156 ICSI cycles using cryopreserved epididymal sperm (ES; n = 77) or testicular sperm (TESE; n = 79). The developmental potential of embryos did not appear to be influenced by the type of surgically retrieved sperm. The average number of blastomeres observed on Day 3 was not different among different groups; 7.5 +/- 1.7 (ES), 7.6 +/- 2.1 (TESE-OA) and 6.5 +/- 2.3 (TESE-NOA). Compaction and blastulation rates, both indicators of paternal genomic activation, were similar in embryos derived from ICSI with ES or TESE from OA as well as NOA men. The only parameter significantly affected in NOA-TESE cases was the fertilization rate. CPR and IR with cryopreserved TESE (TESE-OA 59%, 34%, and TESE-NOA 37%, 20%) were also not statistically different, from that achieved with cryopreserved ES (61% and 39%). Live birth rates also appeared to be independent of sperm type. The 87 clinical pregnancies established using cryopreserved TESE and ES, resulted in the birth of 115 healthy infants. No congenital anomalies were noted.ConclusionZygotic activation seems to be independent of sperm origin and type of azoospermia.