Greta Verheyen

Influence of individual sperm morphology on fertilization, embryo morphology, and pregnancy outcome of intracytoplasmic sperm injection

OBJECTIVE To evaluate the influence of morphology of individual spermatozoa on fertilization and pregnancy outcome. DESIGN Retrospective analysis. SETTING An IVF center in an institutional research environment. PATIENT(S) Fertilization and embryo quality according to individual sperm morphology were analyzed in 662 consecutive ICSI cycles. Pregnancy outcome was evaluated for these cycles and an additional 1005 consecutive ICSI cycles. INTERVENTION(S) ICSI was performed using sperm cells of ejaculated, epididymal, or testicular origin. Observation through an inverted microscope was used to prospectively classify injected sperm cells as normal or morphologically abnormal. MAIN OUTCOME MEASURE(S) Oocyte fertilization, embryo morphology, and pregnancy outcome of unmixed embryo transfers. RESULT(S) Injection of morphologically abnormal spermatozoa (irrespective of origin) resulted in a lower fertilization rate (60.7%) than did injection of morphologically normal spermatozoa (71.7%). Embryo cleavage quality did not differ between groups. Higher pregnancy and implantation rates were obtained in patients with normal sperm morphology (36.7% and 18.7%, respectively) than in those with abnormal sperm morphology (20.2% and 9.6%). CONCLUSION(S) Individual sperm morphology assessed at the moment of ICSI correlated well with fertilization outcome but did not affect embryo development. The implantation rate was lower when only embryos resulting from injection of an abnormal spermatozoon were available.

Intracytoplasmic sperm injection with testicular spermatozoa is less successful in men with nonobstructive azoospermia than in men with obstructive azoospermia

OBJECTIVE To assess the efficiency of intracytoplasmic sperm injection (ICSI) using testicular spermatozoa in cases of nonobstructive azoospermia. DESIGN Retrospective case series. SETTING Tertiary university-based infertility center. PATIENT(S) Overall, 595 couples were included. In 360 couples, the man had normal spermatogenesis. In 118, 85, and 32 couples the man had germ-cell aplasia, maturation arrest, and tubular sclerosis/atrophy, all with focal spermatogenesis present. INTERVENTION(S) We performed 911 ICSI cycles using fresh sperm obtained after testicular biopsies: 306 ICSI cycles used testicular sperm from men with nonobstructive azoospermia, and 605 ICSI cycles used testicular sperm from men with obstructive azoospermia. MAIN OUTCOME MEASURE(S) Fertilization, cleavage, implantation, and pregnancy rates. RESULT(S) Overall, the 2PN fertilization rate was lower in the nonobstructive group: 48.5% vs. 59.7%. There were no differences in in vitro development or in the morphological quality of the embryos. In the nonobstructive group, a total of 718 embryos were transferred (262 transfers) vs. 1,525 embryos in the obstructive group (544 transfers). Both the clinical implantation rate and clinical pregnancy rate per cycle were significantly lower in the nonobstructive group compared with the obstructive group: 8.6% vs. 12.5% and 15.4% vs. 24.0%, respectively. CONCLUSION(S) A statistically significant lower rate of fertilization and pregnancy results from ICSI with testicular sperm from men with nonobstructive azoospermia, compared with men with obstructive azoospermia.

Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics

BACKGROUND Gene expression of cumulus cells (CC) could predict oocyte developmental quality. Knowledge of the genes involved in determining oocyte quality is scanty. The aim was to correlate clinical and biological characteristics during ovarian stimulation with the expression of 10 selected genes in CC. METHODS Sixty-three ICSI patients were stimulated with GnRH-agonist plus highly purified hMG (n = 35) or recombinant FSH (n = 28). Thirteen variables were analyzed: Age, BMI, duration of stimulation, serum concentrations of progesterone, 17beta-estradiol, FSH and LH on day of hCG, Ovarian Response, Oocyte Maturity, 2 pronuclei and three embryo morphology related variables: > or =7 cells, Low Fragmentation, Good Quality Embryos score. Expression of HAS2, VCAN, SDC4, ALCAM, GREM1, PTGS1, PTGS2, DUSP16, SPROUTY4 and RPS6KA2 was analyzed in pooled CC using quantitative PCR, and the relationship to the 13 variables was evaluated by multivariable analysis. RESULTS All 10 genes are expressed at oocyte retrieval, with PTGS1, SPROUTY4, DUSP16 and RPS6KA2 described in human ovary for the first time. The three variables that correlated most often with differential expression were Age, BMI and serum FSH level. Significant correlation was found with Oocyte Maturity (VCAN, P < 0.005), Low Fragmentation (RPS6KA2, P < 0.05), Embryos with > or =7 cells (ALCAM and GREM1, P < 0.05). The expression of the other genes was also correlated to oocyte developmental quality but to a less extent. SDC4, VCAN, GREM1, SPROUTY4 and RPS6KA2 showed gonadotrophin preparation-dependent expression and/or interactions (all P < 0.05). CONCLUSION The expression of ovulation related genes in CC is associated with patient and treatment characteristics, oocyte developmental potential and differs with the type of gonadotrophin used.

Intracytoplasmic sperm injection versus in vitro fertilization: A randomized controlled trial and a meta-analysis of the literature

OBJECTIVE To compare ICSI with IVF using two insemination concentrations in moderate male infertility and to compare these data with other randomized controlled trials (RCTs). DESIGN Prospective, randomized, controlled trial and meta-analysis. SETTING University-based tertiary referral center. PATIENT(S) Seventy-three couples undergoing ART. INTERVENTION(S) In one IVF group a standard insemination concentration of 0.2 x 10(6)/mL was used, whereas in the other group a high insemination concentration (HIC) of 0.8 x 10(6)/mL was used. Each protocol was compared with ICSI on sibling oocytes. MAIN OUTCOME MEASURE(S) Fertilization, fertilization failure, and embryonic development. RESULT(S) The overall fertilization rate was significantly lower after standard IVF than after ICSI: 37.4% vs. 64.3%. Where HIC IVF was used, the overall fertilization rate was not significantly different from that after ICSI: 59.6% vs. 67.6%. Eight randomized controlled trials were selected and reviewed together with the present randomized controlled trial by meta-analysis. The risk ratio for an oocyte to become fertilized was 1.9 (95% confidence interval of 1.4 to 2.5) in favor of ICSI, and 3.1 ICSI cycles may be needed to avoid one complete fertilization failure after conventional IVF (95% CI of 1.7 to 12.4). CONCLUSION(S) The data from this study and the meta-analysis show that ICSI is a more efficient technique in terms of fertilization, but not in comparison with HIC IVF.

Quality of frozen-thawed testicular sperm and its preclinical use for intracytoplasmic sperm injection into in vitro-matured germinal-vesicle stage oocytes.

OBJECTIVE To study the effects of cryopreservation on the quality of human testicular spermatozoa and the efficiency of intracytoplasmic sperm injection (ICSI) with frozen-thawed testicular sperm into metaphase II oocytes in vitro-matured from the germinal-vesicle stage oocyte. DESIGN Preclinical freezing study on supernumarary testicular spermatozoa after ICSI. SETTING Tertiary IVF center coupled with an institutional research environment. PATIENT(S) Twenty-nine patients undergoing excisional testicular biopsy for ICSI. INTERVENTION(S) Isolated testicular spermatozoa were cryopreserved and thawed; frozen-thawed motile testicular spermatozoa were microinjected. MAIN OUTCOME MEASURE(S) Prefreezing and post-thawing motility and viability, survival rate, fertilization rate, cleavage rate, and embryo quality after ICSI. RESULT(S) Mean percentage motility decreased from 21% before freezing to 6% after thawing. Vitality was impaired to a similar extent, decreasing from 68% to 22% (32% recovery rate). Injection of frozen-thawed testicular spermatozoa into in vitro-matured oocytes resulted in a fertilization rate of 50.9%. Cleavage rate was severely impaired. Half of the fertilized oocytes became arrested in the one-cell stage. CONCLUSION(S) Despite the low quality of the fresh testicular spermatozoa, a high percentage of prepared testicular sperm fractions showed survival and motility after the freezing and thawing process. Injection of frozen-thawed testicular sperm into matured oocytes resulted in fertilization rates comparable with these with fresh testicular sperm, but cleavage rates were severely impaired, which might be due to source of oocytes used for ICSI.

NEW BELGIAN EMBRYO TRANSFER POLICY LEADS TO SHARP DECREASE IN MULTIPLE PREGNANCY RATE

Since 1 July 2003, a new transfer policy aiming to reduce multiple pregnancies was brought into law in Belgium. The policy restricts the number of embryos transferred, depending on the patients age and treatment cycle. This study aimed to evaluate the effect of this policy. Two 15-month periods before and after the start of the new law were compared for the following parameters: positive human chorionic gonadotrophin (HCG), clinical pregnancy rate and multiple pregnancy rate according to the age categories defined by the policy: <36, 36-39 and 40-42 years. HCG rates (34.2 and 32.8%) and clinical pregnancy rates (26.2 and 24.0%) per cycle were similar for the two periods. Overall, the multiple pregnancy rate was reduced from 29.1 to 9.5% (all patients) and from 28.9 to 6.2% in women <36 years. Most twins were observed in the third cycle of patients <36 years and in the first three cycles in women of 36-39 years. It can be concluded that a significant decline (P < 0.001) in multiple pregnancies was mainly observed in patients <36 years of age. Clinical pregnancy rates were not compromised by the new law. Elective single embryo transfer should be considered more seriously for women 36-39 years of age.

Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients

BACKGROUND Cumulus cell (CC) gene expression is suggested as a non-invasive analysis method to predict oocyte competence. There are, however, important between-patient differences in CC gene expression. These can be compensated when expression results are combined with patient and cycle characteristics using a multiple regression analysis model. METHODS From ICSI patients stimulated with GnRH antagonist and recombinant FSH (n= 25) or GnRH agonist and highly purified menotrophin (n= 20), CC were collected and oocytes were individually fertilized and cultured. CC were analyzed for the expression of Syndecan 4 (SDC4), Prostaglandin-endoperoxide synthase 2 (PTGS2), Versican (VCAN), Activated leukocyte cell adhesion molecule, Gremlin 1, transient receptor potential cation channel, subfamily M, member 7 (TRPM7), Calmodulin 2 and Inositol 1,4,5-trisphosphate 3-kinase A (ITPKA) using quantitative PCR. Results were analyzed in relation to the stimulation protocol. Within-patient variation in gene expression was related to oocyte maturity and developmental potential. Models predictive for normal embryo or blastocyst development and pregnancy in single embryo transfer cycles were developed. RESULTS Mature oocytes have higher PTGS2 and lower VCAN expression in their cumulus. All genes except VCAN had a positive correlation with good embryo or blastocyst morphology and were used to develop predictive models for embryo or blastocyst development (P< 0.01). Specific models were obtained for the two stimulation protocols. In both groups, better cleavage-stage embryo prediction relied on TRPM7 and ITPKA expression and pregnancy prediction relied on SDC4 and VCAN expression. In the current data set, the use of CC expression for pregnancy prediction resulted in a sensitivity of >70% and a specificity of >90%. CONCLUSIONS Multivariable models based on CC gene expression can be used to predict embryo development and pregnancy.

The use of testicular sperm for intracytoplasmic sperm injection in patients with necrozoospermia.

OBJECTIVE To investigate whether intracytoplasmic sperm injection (ICSI) using testicular spermatozoa from necrozoospermic patients results in acceptable fertilization and transfer rates. DESIGN Retrospective clinical study. SETTING Tertiary referral center. PATIENTS Five patients presenting consistently with 100% dead spermatozoa in their ejaculates. INTERVENTIONS In seven treatment cycles, an open testicular biopsy was performed to increase the chances for retrieving viable spermatozoa which were used for ICSI. MAIN OUTCOME MEASURES Sperm recovery, fertilization, and transfer rates. RESULTS Testicular sperm were recovered in all treatment cycles and fertilization occurred in six of seven cycles. Overall normal fertilization and transfer rates were 67% and 71%, respectively. One live birth was obtained after five ETs. CONCLUSION We recommend testicular sperm recovery for ICSI in patients who invariably or occasionally present with absolute necrozoospermia.

An improved treatment procedure for testicular biopsy specimens offers more efficient sperm recovery: Case series

OBJECTIVE To report an improved sperm recovery procedure from testicular biopsy specimens for intracytoplasmic sperm injection (ICSI). DESIGN Case series and controlled study. SETTING Procedures were performed in a tertiary IVF center. PATIENT(S) Nonobstructive azoospermic cases (15 patients) and obstructive azoospermic cases (5 patients). INTERVENTION(S) Intracytoplasmic sperm injection was carried out using testicular sperm isolated from a testicular biopsy specimen either with or without erythrocyte lysing buffer treatment. MAIN OUTCOME MEASURE(S) The time required to collect spermatozoa and the intactness and fertilization and developmental rates of oocytes. RESULT(S) In 7 of the 15 nonobstructive cases, it was possible to perform ICSI when, after shredding of the testicular tissue, no (or virtually no) sperm were present. There was no difference in the fertilization rates (83% and 68%) and developmental rates (87% and 89%) of the 54 sibling oocytes from another 5 patients in whom ICSI was carried out with sperm either treated or not treated with erythrocyte lysing buffer. CONCLUSION(S) Erythrocyte lysing buffer treatment of testicular biopsy specimens enhances the efficiency of sperm collection in those cycles in which spermatozoa are present and does not affect fertilization and embryo development.

Obstetric outcome in donor oocyte pregnancies: a matched-pair analysis

BackgroundTo investigate the obstetrical and perinatal impact of oocyte donation, a cohort of women who conceived after OD was compared with a matched control group of women who became pregnant through in vitro fertilisation with autologous oocytes (AO).MethodsA matched-pair analysis has been performed at the Centre for Reproductive Medicine of the UZ Brussel, Dutch speaking Free University of Brussel. A total of 410 pregnancies resulted in birth beyond 20 weeks of gestation occurring over a period of 10 years, including 205 oocyte donation pregnancies and 205 ICSI pregnancies with autologous oocytes (AO). Patients in the OD group were matched on a one-to-one basis with the AO group in terms of age, ethnicity, parity and plurality. Matched groups were compared using paired t-tests for continuous variables and McNemar test for categorical variables. A conditional logistic regression analyses was performed adjusting for paternal age, age of the oocyte donor, number of embryos transferred, and singleton/twin pregnancy.ResultsOocyte donation was associated with an increased risk of pregnancy induced hypertension (PIH) (matched OR: 1.502 CI: 1.024-2.204), and first trimester bleeding (matched OR: 1.493 CI: 1.036-2.15). No differences were observed between the two matched groups with regard to gestational age, mean birth weight and length, head circumference and Apgar scores.ConclusionsOocyte donation is associated with an increased risk for PIH and first trimester bleeding independent of the recipients’ age, parity and plurality, and independent of the age of the donor or the partner. However, oocyte donation has no impact on the overall perinatal outcome.