O. Bern

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.

CLINICAL ASSISTED REPRODUCTION: Improvement of IVF Outcome in Poor Responders by Discontinuation of GnRH Analogue During the Gonadotropin Stimulation Phase—A Function of Improved Embryo Quality

Purpose: To assess the efficacy of a protocol involving the discontinuation of the GnRH analogue at the mid-phase of ovarian stimulation for IVF in patients with a previous poor response.Methods: Prospective case-control evaluation compared with same patients previous performance. Thirty-six patients enrolled in an IVF program were treated in two consecutive cycles. The first with a standardized protocol utilizing mid-luteal administration of Nafarelin (N) 600 mcg/d continued throughout the stimulation phase with human menopausal gonadotropin (hMG) until follicles of 20 mm were identified by transvaginal ultrasound (Standard group). Patients with a poor response in the Standard cycle were treated in the subsequent cycle with N and hMG initially in a similar manner, then N was stopped after 5 days of hMG stimulation (N-stop group). All clinical and laboratory aspects of treatment were done in a similar fashion in both cycles, each patient acting as her own control.Results: Results were analyzed by paired t test. The change in each parameter in the N-stop cycle was expressed as the percent change as compared with the standard protocol cycle for each patient. Peak estradiol (E2) and number of aspirated oocytes were increased in the N-stop cycle (+16.9% and +28%, respectively), but insignificantly so. The percent of cleaving embryos was significantly increased by 27.9% (p = 0.03) in the N-stop cycle, as embryo morphology was improved by 22% (p = 0.02). The efficacy of gonadotropin treatment was enhanced in the N-stop cycle, as expressed by a 32.5% increase in oocytes retrieved per hMG ampoule administered (p = 0.04). Three cycles of 36 were cancelled during the N-stop cycle, whereas only one was cancelled in the standard protocol cycle. Of the 36 patients, 7 conceived in the N-stop protocol and 5 are ongoing pregnancies.Conclusion: Discontinuation of GnRH-a during ovarian stimulation for IVF has a beneficial, but not statistically significant, effect on both E2 and oocyte production. Embryo cleavage rates and morphology were significantly improved, this may be due to improved oocyte quality, which may have been responsible for achieving pregnancies. The efficacy of gonadotropin treatment was enhanced when GnRH-a was discontinued. These results hint that GnRH-a may have a direct negative effect on folliculogenesis and oocytes, which is apparent especially in poor responder patients.

Presence of IL-18 in testicular tissue of fertile and infertile men.

Recently, IL‐18 was identified in human testes. Moreover, an inverse correlation was found between the levels of IL‐18 and the number and motility of spermatozoa. We examined the presence of IL‐18 protein in normal and impaired spermatogenesis. Testicular tissue specimens were taken from 25 nonobstructive azoospermic patients undergoing testicular sperm extraction and from autopsies of three healthy controls. The presence of IL‐18 in human testicular cells was examined by immunohistochemical staining of paraffin‐embedded sections, using a specific antibody for human IL‐18. In testicular tissue of healthy controls as well as in study cases, presence of IL‐18 was identified in somatic, mitotic, meiotic and post‐meiotic cells in correlation with their presence. In all patients, Leydig cells were less intensively stained. Mitotic cells were immunostained in the control group and less intensively in hypospermatogenesis and maturation arrest subgroups. Primary spermatocytes were in general most efficiently stained. The expression of IL‐18 mRNA (as examined by real‐time PCR analysis) showed significantly lower expression in testicular tissues with impaired spermatogenesis when compared to normal tissues. We report the first study demonstrating the presence of IL‐18 in human testicular tissue at the protein level. The presence of this cytokine in somatic as well as in different types of germ cells may suggest its involvement in the regulation of the spermatogenic process and steroidogenesis under physiological and pathological conditions.

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.

Preimplantation genetic diagnosis (PGD) for SHOX-related haploinsufficiency in conjunction with trisomy 21 detection by molecular analysis

PurposeDevelopment of a molecular PGD protocol for a male with an X-linked deletion in the SHOX gene region, located in the pseudoautosomal region of the X/Y chromosomes. Due to excessive recombination in this region, the deletion can be found in male offspring.MethodsWe developed a 13 marker multiplex fluorescent PCR protocol: 3 markers within the deleted SHOX region, 5 flanking markers, 3 informative markers on chromosome 21 (advanced maternal age) and 2 markers for sex determination.ResultsOf four embryos, two wild type males, diploid for chromosome 21 were transferred resulting in twin boys. One embryo was an affected female and another embryo was Turner. Amniocentesis confirmed the implanted embryos were males (46XY), with no recombinations.ConclusionsWhile many X-linked disorders can be analyzed by sexing, genes located in the pseudoautosomal regions have high XY recombination rates, requiring multiple markers to enable an accurate diagnosis.

Fertilizing Capability of Frozen-Thawed Immotile Sperm

The introduction of intracytoplasmic sperm injection (ICSI) for severe male cases has made it possible to treat patients with azoospermia, in whom sperm is achieved from the epididymis and the testis (1–3). In some of the treated male patients, especially those with non-obstructive azoospermia, mainly immotile spermatozoa are available. Because it is assumed that the fertilizing potential of testicular sperm is lower (4) and motility is an important predictive factor for successful fertilization (5) it should be questioned whether ICSI perfomed with immotile sperm is justified.