Tracking of oocyte dysmorphisms for ICSI patients may prove relevant to the outcome in subsequent patient cycles.

BACKGROUND: We determined whether oocyte dysmorphisms, especially repetition of specific dysmorphisms from cycle to cycle, had a prognostic impact on intracytoplasmic sperm injection (ICSI) outcome. METHODS: ICSI patients (n = 67) were grouped as follows: group 1 >50% phenotypically dysmorphic oocytes per cohort (cytoplasmic and extra-cytoplasmic dysmorphisms) with no repetition of a specific dysmorphism from cycle one to cycle two (36 cycles and 274 oocytes); group 2 >50% dysmorphic oocytes per cohort and repetition of the same dysmorphism from cycle one to cycle two (32 cycles and 313 oocytes); group 3 (control) <30% dysmorphic oocytes (33 cycles and 378 oocytes). RESULTS: In group 2 (repetitive), 47% of oocytes were observed to have organelle clustering versus 20.5% in group 1 and 17.3% in group 3 (P < 0.001). There was no difference between the groups in fertilization rates, cleavage rates or embryo quality. Embryos derived from normal oocytes were transferred in each group (57, 33 and 72% respectively). The clinical pregnancy and implantation rates in group 2 (3.1 and 1.7% respectively) were lower (P < 0.01, P = 0.005) than both group 1 (28 and 15% respectively) and group 3 (45.5 and 26.5% respectively). CONCLUSIONS: The low implantation rate in group 2, even though 33% of transferred embryos were derived from morphologically normal oocytes, suggests that repetitive organelle clustering may be associated with an underlying adverse factor affecting the entire follicular cohort.

The choice of embryo transfer catheter affects embryo implantation after IVF.

OBJECTIVE: Comparison of two transfer catheters in an IVF program. DESIGN: Prospective, randomized clinical study. SETTING: A private tertiary care center for ART. PATIENT(S): 66 patients < 38 years of age undergoing IVF and/or ICSI. INTERVENTION(S): Patients were randomly assigned to undergo ET using the Tomcat catheter (n = 32) or the TDT catheter (n = 34). MAIN OUTCOME MEASURE(S): Primary outcome measures were implantation and pregnancy rates. Secondary outcome measures were contamination with blood and/or mucus on the tip of the catheter, cramping or patient discomfort, and time required to complete ET. RESULT(S): Use of the Tomcat catheter resulted in significantly higher implantation (25.2% vs. 8.4%) and clinical pregnancy rates (47% vs. 14.7%) compared with the TDT catheter. All secondary outcome measures were similar for both catheters. CONCLUSION(S): The choice of ET catheter may affect the success of IVF-ET cycles. Use of the Tomcat catheter compared with the TDT catheter seems to result in significantly better efficiency of the ET procedure and is more cost effective.

Variability in the expression of trophectodermal markers beta-human chorionic gonadotrophin, human leukocyte antigen-G and pregnancy specific beta-1 glycoprotein by the human blastocyst.

Improved culture conditions that support the development of human embryos to the blastocyst stage in vitro led to the prospect of blastocyst transfer to increase pregnancy rates. Thus, there is a need for characterization of possible biochemical markers able to predict the implantation potential of human blastocysts. In this study, the expression of three placental markers that are expressed prior to implantation, beta-human chorionic gonadotrophin (HCG), human leukocyte antigen (HLA)-G and pregnancy specific beta-1 glycoprotein (SP-1), was investigated. beta-HCG transcript could be detected as early as the two-cell stage, which is one to two cleavage divisions earlier than previously reported. Both beta-HCG and HLA-G transcripts could be detected in the majority of blastocysts, but their levels were highly variable. No association could be found between the amount of transcript for these genes, total cell number or cell death rate. Interestingly, there was a highly positive correlation between accumulation of beta-HCG and HLA-G transcripts. SP-1 protein concentrations were assessed in the culture medium of blastocysts using enzyme-linked immunosorbent assay. There was a significant positive correlation between SP-1 concentrations and blastocyst cell numbers. Moreover, synthetic oviductal medium enriched with potassium resulted in an SP-1 concentration twice as high as that observed using human tubal fluid medium. These data suggest that SP-1 may be used to select blastocysts with higher cell number, possibly resulting in higher pregnancy rates.

DNA damage in round spermatids of mice with a targeted disruption of the Pp1cgamma gene and in testicular biopsies of patients with non-obstructive azoospermia.

Non-obstructive azoospermia accounts for a considerable proportion of male factor infertility. Current therapies for treatment of this kind of infertility include procedures such as intracytoplasmic sperm injection (ICSI), round spermatid injection (ROSI), round spermatid nucleus injection (ROSNI) and elongated spermatid injection (ELSI). All involve injection of haploid germ cells retrieved from testicular biopsies into recipient oocytes. We have investigated a mouse model of azoospermia for quality of haploid germ cell genomes, based on 4,6-diamidino-2-phenylindole (DAPI)/TdT-mediated dUTP nick-end labelling (TUNEL) labelling. The mouse model, a targeted mutation in the protein phosphatase 1cg gene, results in severe depletion of haploid germ cells from the round spermatid stage on. Mice homozygous for the mutation are completely infertile, and produce only the occasional spermatozoon. Spermatozoa and round spermatids retrieved from either the epididymides or the testes of mutant mice displayed very high rates of DNA fragmentation. In contrast, similar cells retrieved from heterozygous or wild-type littermates displayed low levels of DNA fragmentation. In some cases, the high rates of DNA fragmentation in mutant cells could be lowered by inclusion of antioxidants in the retrieval media. High rates of DNA fragmentation were also observed in round spermatids retrieved from testicular biospies of human patients with non-obstructive azoospermia. These results suggest that one of the features of the pathology associated with azoospermia is fragmented DNA in haploid germ cells. This raises questions about the suitability of using these cells for fertility treatment.

Intracytoplasmic sperm injection after follicle stimulation with highly purified human follicle-stimulating hormone compared with human menopausal gonadotropin.

PURPOSE: Our purpose was to compare oocyte nuclear maturation and embryo quality after pituitary down-regulation and ovarian stimulation with highly purified follicle-stimulating hormone (FSH) or human menopausal gonadotropin (HMG). METHODS: Fifty-five patients 37 years of age or younger who were undergoing in vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) were evaluated retrospectively. In all cases, male factor was the only indication for treatment, with no female-related factors identified. Following pituitary down-regulation, patients were stimulated with hMG (n = 20) or highly purified FSH (n = 35). Main outcome measures included ovarian response to stimulation, oocyte maturity, and ICSI fertilization results. Secondary outcome measures included pregnancy rates and outcome. RESULTS: The ovarian response to stimulation was similar for the two groups, as were the percentage of metaphase II oocytes, fertilization and cleavage rates, and number and quality of transferred and cryopreserved embryos. Cycle outcome was comparable. CONCLUSIONS: In normogonadotropic subjects, monocomponent therapy with highly purified FSH is as effective as hMG in stimulating ovarian follicular development, synchronization of oocyte maturation, and IVF-ICSI outcome. Our findings support the conclusion that the luteinizing hormone component in the stimulation protocol is unnecessary.

Fresh and frozen epididymal sperm yield comparable pregnancy rates for intracytoplasmic sperm injection.

To determine if the use of fresh epididymal sperm is superior to frozen-thawed epididymal sperm for intracytoplasmic sperm injection, the authors reviewed the charts on all couples undergoing intracytoplasmic sperm injection at an academic center, using microsurgically aspirated epididymal sperm. Forty-nine couples undergoing intracytoplasmic sperm injection for male factor infertility, due to congenital absence of vas deferens or irreparable post-testicular obstruction were studied. The following parameters were measured: (1) fertilization rate per oocyte injected (two pronuclei at 24 h), (2) chemical pregnancy rate (two consecutively elevated serum b-hCG levels, and (3) clinical pregnancy rate (sonographic identification of fetal heart rate). Fertilization rates were 51 and 41%, chemical pregnancy rates were 27 and 30%, and clinical pregnancy rates were 19 and 27% in the fresh epididymal compared to the frozen epididymal sperm. This study shows no significant difference in outcomes using fresh or frozen epididymal sperm for intracytoplasmic sperm injection. Frozen-thawed sperm guarantees availability of sperm prior to oocyte retrieval.

Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection.

OBJECTIVE: To determine the incidence of DNA fragmentation in human sperm used for intracytoplasmic sperm injection (ICSI) and to correlate any detected DNA damage with semen analysis parameters and fertilization rates in ICSI. DESIGN: Descriptive and correlational clinical study. SETTING: Tertiary care fertility clinic. PATIENT(S): A total of 150 semen samples was collected from men in the ICSI program. INTERVENTION(S): For each sample, sperm wash and swim-up were performed, and the percentage of recovered sperm with DNA fragmentation was determined with the use of terminal transferase-mediated deoxyuridine triphosphate-biotin end labeling. MAIN OUTCOME MEASURE(S): The percentage of sperm with DNA fragmentation was correlated with semen analysis parameters and ICSI fertilization rates. RESULTS(S): The mean (+/- SD) percentage of sperm with fragmented DNA was 14.5% +/- 1.5% and ranged from 0.5% to 75%. A significant negative association was found between the percentage of sperm with DNA fragmentation and the ICSI fertilization rate. We also observed that the motility and morphology of the ejaculated sperm were correlated negatively with the percentage of DNA fragmentation in the washed sperm recovered by the swim-up technique. CONCLUSION(S): Our results suggest that when poor-quality semen samples are used for ICSI, there is a greater likelihood that some sperm selected for injection, despite appearing normal, contain fragmented DNA. Whether sperm DNA damage may contribute to failure of pronuclear formation and embryo development in some apparently unfertilized ICSI oocytes is unclear.

Intracytoplasmic sperm injection for treatment of infertility due to acrosomal enzyme deficiency.

OBJECTIVE: To determine whether absence of fertilization in IVF associated with an acrosomal enzyme defect (hyaluronidase deficiency) results from a simple mechanical block to sperm penetration or from a more serious sperm abnormality. DESIGN: Nonrandomized, prospective study. SETTING: Toronto Center for Advanced Reproductive Technology, a tertiary referral center for infertility associated with The University of Toronto. PATIENTS: One hundred twenty-two couples about to undergo intracytoplasmic sperm injection (ICSI) were selected. Thirty-six of the studied couples had failed to fertilize in prior IVF cycles. INTERVENTIONS: Hyaluronidase activity was measured in the semen samples provided for ICSI using a zymogenic assay. Intracytoplasmic sperm injection was performed in all couples using standard techniques. RESULTS: Forty-eight of 122 semen samples had poor of absent semen hyaluronidase activity. All 48 samples resulted in successful fertilization with ICSI in the present study. The average fertilization rate per oocyte was 59.43% in couples in whom the partner had low semen hyaluronidase activity and 55.85% in whom the male had normal hyaluronidase activity. The ET rate per cycle was 100% and 95% and pregnancy rates per cycles were 26% and 25% in cycles with poor and normal semen hyaluronidase activity, respectively. Unlike routine IVF, no statistical correlation was found between semen hyaluronidase activity and the fertilization rate in ICSI. CONCLUSION: Our results indicates that semen hyaluronidase deficiency is associated with a simple mechanical block to fertilization. In addition, the measurement of semen hyaluronidase activity can provide a reliable means for selecting couples who would benefit from ICSI.

The hypo-osmotic swelling test for selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenozoospermia.

OBJECTIVE: To determine the ability of the hypo-osmotic swelling test to select viable sperm from nonmotile sperm samples for intracytoplasmic sperm injection (ICSI). DESIGN: Nonrandomized, sequential comparative study. PATIENTS: Thirteen couples enrolled in our ICSI program had 16 cycles in which sperm preparations with 0% motility were obtained. Five cycles used cryopreserved epididymal sperm with complete asthenozoospermia. INTERVENTIONS: In eight cycles, the semen samples were washed through a Percoll gradient and sperm were selected randomly for ICSI. In another eight cycles, the washed sperm were placed in a hypo-osmotic solution (75 mM fructose; 25 mM sodium citrate dihydrate) and the sperm with curled tails taken up with the microinjection needle, rinsed, and used ICSI. MAIN OUTCOME MEASURES: Fertilization rate per oocyte injected as determined by the presence of two pronuclei at 18 hours after retrieval and embryo cleavage rate per oocyte injected at 48 hours after retrieval. RESULTS: With random sperm injection, the fertilization and cleavage rates were 26% and 23%, respectively. In contrast, after injection of sperm selected using the hypo-osmotic swelling test, fertilization and cleavage rates were significantly greater (43% and 39%, respectively). There were three pregnancies in the eight cycles with the hypo-osmotic swelling test-selected sperm, including two from frozen epididymal sperm. CONCLUSION: Based on these preliminary observations, we believe that the hypo-osmotic swelling test will prove to be valuable for increasing fertilization and cleavage rates and pregnancy rates in ICSI cycles where no motile sperm are recovered.

Type of stimulation protocol affects oocyte maturity, fertilization rate, and cleavage rate after intracytoplasmic sperm injection.

OBJECTIVE: To compare oocyte maturity, fertilization rate and cleavage rate after a short and long GnRH agonist (GnRH-a) stimulation protocol and intracytoplasmic sperm injection (ICSI). DESIGN: Retrospective study of 34 sequential ICSI cycles stimulated with a short or long GnRH-a protocol. SETTING: A university-based tertiary care center for assisted reproductive treatment. RESULTS: Significantly more oocytes were mature (metaphase II) after a long GnRH-a protocol then after a short GnRH-a protocol (25.6% and 80.8%, respectively). The long protocol resulted in more cleaving embryos (36/152 versus 9/132) and more cycles of ET (12/17 versus 5/17) than the short group. CONCLUSION: A greater percentage of mature oocytes results from ovarian stimulation with a long GnRH-a protocol than a short GnRH-a protocol. Maturity could be assessed accurately after cumulus stripping that is required before ICSI. Fertilization rate and cleavage rate with ICSI was superior after a long GnRH-a stimulation protocol for superovulation.