D. Komarovsky

In vitro maturation of human germinal vesicle-stage oocytes: role of epidermal growth factor-like growth factors in the culture medium

BACKGROUND In vitro maturation (IVM) of oocytes is a promising technique to reduce the costs and avert the side effects of gonadotrophin stimulation for IVF. The pregnancy rates from oocytes matured in vitro are still lower than those of in vivo stimulation cycles, indicating that optimization of IVM remains a challenge. Recently, it was demonstrated that LH exerts its action on ovulation, at least in part, through stimulation of the production of the epidermal growth factor family members amphiregulin (Areg) and epiregulin (Ereg) in pre-ovulatory follicles, and they, in turn, serve as paracrine mediators of LH. We aimed to investigate the effect of supplementation of the medium with Areg and Ereg on the maturation rate of immature oocytes. METHODS A total of 105 sibling human germinal vesicle (GV) oocytes obtained after gonadotrophin stimulation were cultured in a complex defined medium either with or without supplemented recombinant human Areg (75 ng/ml) and Ereg (75 ng/ml) for 24 h. RESULTS Significantly more oocytes reached the metaphase II stage at 24 h in media supplemented with Areg and Ereg (75.5 versus 36.5%, P < 0.001). In vitro matured oocytes retrieved from the two subgroups had no statistically significant difference in fertilization and cleavage rates or morphology scores. Overall, a significantly higher number of Day 2 (52.8 versus 26.9% P < 0.01) and Day 3 (45.2 versus 23%, P < 0.05) embryos originated from GV oocytes cultured in the Areg- and Ereg-enriched medium. CONCLUSIONS Supplementation of the maturation medium with Areg and Ereg improves the maturation of human GV oocytes in vitro.

Intracytoplasmic sperm injection outcome of ejaculated versus extracted testicular spermatozoa in cryptozoospermic men

OBJECTIVE To compare intracytoplasmic sperm injection (ICSI) outcome of patients with cryptozoospermia after use of ejaculated versus testicular sperm in different cycles of the same patients. DESIGN Retrospective cohort study. SETTING University-affiliated infertility center. PATIENT(S) A total of 17 patients with cryptozoospermia who underwent a total of 116 ICSI cycles. INTERVENTION(S) The patients initially underwent several ICSI cycles using ejaculated sperm (n = 68, 58.6%) that were followed by ICSI cycles using testicular sperm (n = 48, 41.4%). MAIN OUTCOME MEASURE(S) Fertilization rate, pregnancy rate (PR). RESULT(S) There were no significant differences in fertilization rates between the two subgroups. A comparison between testicular sperm extraction (TESE) versus ejaculated sperm cycles revealed significantly higher implantation rate (20.7% vs. 5.7%), higher PR (42.5% vs. 15.1%), and higher take home baby rate (27.5% vs. 9.4%). A multivariable logistic regression analysis showed three significant predictors for pregnancy, namely the use of testicular sperm (odds ratio [OR] 5.1, 95% confidence interval [95% CI] 1.8-14.8), use of motile sperm (OR 12.9, 95% CI 2.1-79.1), and female age (OR 0.83, 95% CI 0.7-0.9). CONCLUSION(S) Testicular sperm extraction is justified in patients with cryptozoospermia who fail to conceive by ICSI using ejaculated spermatozoa, as it offers higher PR.

Computerized cell-scanning system for evaluating human spermatogenesis in non-obstructive azoospermic patients

There may be incompatibility between testicular histopathological evaluation and testicular sperm extraction (TESE) outcome. Assessment for sperm presence and different pathological disturbances of non-obstructive azoospermia (NOA) remains challenging. An assay for maximal sampling and accurate identification of testicular cells from NOA patients undergoing TESE and autopsied fertile controls was developed. Testicular cells stained and scanned automatically for morphology underwent fluorescence in-situ hybridization using centromeric probes for chromosomes X, Y and 18 after destaining. Cells were automatically classified according to ploidy, and ratios of haploid cells and autosomal (18) and sex-chromosome bivalent rates were calculated. Identification of testicular cells in suspension enabled prediction of spermatogenesis in seven of eight Sertoli-cell-only syndrome patients. Haploid/diploid cell ratios were 67.6:32.2 for controls and 9.6:90.4 for patients. Both autosomal (18) and sex-chromosome bivalents were present in patients (4.1 ± 5.82%) and controls (19.7 ± 8.95%). Few tetraploid pachytene spermatocytes were observed. More secondary spermatocytes with NOA showed two distinct signals for chromosome 18 (27.9 ± 32.69%) compared with controls (0.4 ± 0.35%). The computerized cell-scanning system enables simultaneous application of morphology and chromosome analysis of testicular cells, which enhance assessing different pathological disturbances and estimating the likelihood of a successful second TESE procedure.

Reproductive Performance of Patients Undergoing Intracytoplasmic Sperm Injection with 100% Implantation Rate

Purpose: To characterize the differences between twomatched groups of patients treated by ICSI: those pregnantafter all embryos transferred implanted (100% implantationrate) compared with nonpregnant patients. Methods: Twenty-one patients in whom one transferredembryo achieved a singleton pregnancy (group A) and 21pregnant patients to whom two or three embryos were transferredand achieved 11 twin and 10 triplet pregnancies(group B) compared with matched nonpregnant patients(group C and D, respectively). Results: The singleton pregnant patients were significantlyolder than the twin and triplet pregnancy patients. Althougha similar number of human menopausal gonadotropinampules were used in the singleton compared with the twinsand triplets a significantly lower number of oocytes andembryos were achieved at lower levels of estradiol on thehuman chorionic gonadotropin day in the former than inthe latter respectively. No difference was found between thepregnant women and their nonpregnant controls in any ofthe mentioned parameters. Good embryo morphology wasfound in 86% of the embryos in group A compared with62% in group C (P = 0.08) and 92% in group B comparedwith 66% in group D (P < 0.002). Conclusions: The only parameter in which pregnant patientswith 100% implantation rate differ from their nonpregnantcontrols was embryo quality.