Ploidy of spermatogenic cells of men with non-mosaic Klinefelter's syndrome as measured by a computerized cell scanning system.

PURPOSE: This study aims to characterize the origin of testicular post-meiotic cells in non-mosaic Klinefelter's syndrome (KS). METHODS: The study included testicular tissue specimens from 11 non-mosaic KS patients, with (6 positive) and without (5 negative) spermatozoa presence. The obtained testicular cells were affixed and stained for morphology followed by fluorescence in situ hybridization (FISH) for centromeric probes X, Y, and 18. We used a computerized automated cell scanning system that enables simultaneous viewing of morphology and FISH in the same cell. RESULTS: A total of 12,387 cells from the positive cases, 11,991 cells from the negative cases, and 1,711 cells from the controls were analyzed. The majority of spermatogonia were 47, XXY in both the positive and negative KS cases (88.9 ± 4.76 % and 90.6 ± 4.58 %) as were primary spermatocytes (76.8 ± 8.14 % and 79.6 ± 7.30 %). The respective rates of secondary spermatocytes and post-meiotic cells (round, elongating spermatids and sperm cells) were 1.1 ± 1.39 % in the positive cases, 2.9 ± 3.33 % in the negative cases, compared to 67.6 ± 6.22 % in the controls (P < 0.02). Pairing of both 18 and XY homologous chromosomes in 46,XY primary spermatocytes was 2.5 ± 2.31 % and 3.4 ± 2.39 %, respectively, compared to 19.8 ± 8.95 % in the control group (P < 0.02) and in 47,XXY primary spermatocytes in 2.4 ± 3.8 % in the positive group and 3.2 ± 2.26 % in the negative group. CONCLUSIONS: This study presents data to indicate that the majority of primary spermatocytes in the testes of non-mosaic KS patients are 47,XXY and could possibly develop into post-meiotic cells.

The effect of two distinct levels of oxygen concentration on embryo development in a sibling oocyte study.

PURPOSE: This prospective randomized study used sibling oocytes of 258 women with ≥8 oocytes to compare the effect of 5 % O(2) versus 20 % O(2) concentrations on embryo development and clinical outcome. METHODS: Oocytes of each case were divided between incubators with either 5 % or 20 % O(2) concentration. Outcome measures were fertilization, cleavage, embryo quality, blastocyst formation, and implantation, pregnancy and live birth rates. RESULTS: Fertilization and cleavage rates were similar in both groups. The 5 % O(2) group had significantly more blastomeres (P < 0.05) and more top-quality embryos on day 3 (P < 0.02), as well as significantly more available embryos for transfer (31.6 % vs. 23.1 % for the 20 % O(2) group; P < 0.0001). There were significantly more cycles with good embryos in the 5 % group (76/258) than in the 20 % group (38/258) (P < 0.0001). Implantation and pregnancy rates were significantly higher for 5 % O(2) embryos (P < 0.03 and P < 0.05, respectively). Live birth rates per embryo transfer were 34.2 % and 15.8 %, respectively, P < 0.05. CONCLUSIONS: Implantation, pregnancy and live birth rates are higher, and more good quality embryos are available for transfer and freezing with reduced rather than with atmospheric oxygen concentrations during embryo incubation.

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.

PURPOSE: Development 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. METHODS: We 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. RESULTS: Of 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. CONCLUSIONS: While 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.

Can pregnancy rate be improved in gonadotropin-releasing hormone (GnRH) antagonist cycles by administering GnRH agonist before oocyte retrieval? A prospective, randomized study.

OBJECTIVE: To examine whether the addition of one dose of preovulatory GnRH agonist could improve implantation and pregnancy rates in GnRH antagonist IVF cycles. DESIGN: Prospective, randomized clinical trial. SETTING: University-affiliated IVF and infertility unit. PATIENT(S): Two hundred twenty-one patients intended for GnRH antagonist protocol IVF. INTERVENTION(S): Patients were prospectively randomized to two groups. The control group received hCG (5,000 U) 34 hours before oocyte aspiration, and the study group received triptorelin (0.2 mg SC) in addition to hCG. All other treatment parameters were identical. MAIN OUTCOME MEASURE(S): Oocyte pick-up day serum levels of E(2), P, LH, and FSH and implantation and pregnancy rates per started cycle and per completed cycle. RESULT(S): A total of 200 ET cycles were carried out: 97 in the study group and 103 in the control group. None of the cycle parameters of the study or control groups differed, excepting mean oocyte pick-up day FSH (11.26 IU/L [95% confidence interval (CI) 9.88-12.52] vs. 6.27 IU/L [95% CI 5.76-8.77]) and LH levels (5.19 IU/L [95% CI 4.47-5.9] vs. 3.28 IU/L [95% CI 2.22-4.18]). The implantation rate was 19.9% (52 of 261) for the study group and 13.9% (35 of 251) for the control group. The pregnancy rate in completed cycles and the ongoing pregnancy rate per ET were significantly higher in the study group than in the control group: 29.1% (30 of 103) in the control group and 44.3% (43 of 97) in the study group, and 22.3% (23 of 103) and 36.1% (35 of 97), respectively. However, the improvement in pregnancy rate per started cycle did not reach statistical significance (40.9% vs. 28.3%). CONCLUSIONS: The administration of triptorelin (0.2 mg) at the time of hCG administration in GnRH antagonist IVF cycles significantly improved overall and ongoing pregnancy rates in completed cycles but not in all started cycles. It is possible that this was achieved owing to an endometrial GnRH receptor effect, which should also be examined by direct endometrial studies.

Favorable influence of local injury to the endometrium in intracytoplasmic sperm injection patients with high-order implantation failure.

Local injury to the endometrium prior to controlled ovarian stimulation may considerably improve implantation rates and pregnancy outcomes in intracytoplasmic sperm injection patients with high-order implantation failure (> or =4 IVF trials and > or =12 transferred embryos).

Insulin resistance in patients with polycystic ovary syndrome is associated with elevated plasma homocysteine.

BACKGROUND: Elevated levels of plasma homocysteine have recently been implicated as a significant risk factor for cardiovascular disease, pre-eclampsia, and recurrent pregnancy loss, and have been found to be associated with insulin resistance in a number of clinical situations. We examined the relationship between plasma homocysteine and insulin resistance in patients with polycystic ovary syndrome (PCOS). METHODS: A total of 155 infertile patients with PCOS as defined by clinical, biochemical and ultrasound criteria were screened for insulin resistance utilizing single-sample fasting insulin and glucose measurement, calculated by glucose:insulin ratio or homeostasis model assessment (HOMA) index. Total plasma homocysteine was measured by fluorescence polarization immunoassay. One hundred normo-ovulatory women with normal ovaries being treated for other infertility diagnoses served as a control group. RESULTS: Insulin resistance was found in the majority of PCOS patients: -53.5% (83/155), 60.6% (94/155) and 65.8% (102/155), when defined by fasting insulin, glucose:insulin ratio, or logHOMA respectively. Mean plasma homocysteine in the PCOS group was significantly higher than in the normal ovary group (11.5 +/- 7.4 versus 7.4 +/- 2.1 micromol/l, P < 0.001). Insulin-resistant PCOS patients had significantly higher plasma homocysteine (12.4 +/- 8.4 micromol/l) than non-insulin-resistant PCOS patients (9.6 +/- 4.4 micromol/l) regardless of body mass index (P = 0.003 by groups, P = 0.005 by correlation of single samples). Thirty-four per cent (53/155) of the PCO patients had homocysteine values >95th percentile of the controls (11.0 micromol/l, P < 0.0001). Statistically significant correlations were found between all insulin resistance indices and homocysteine levels. Multiple logistic regression defined insulin resistance as the major factor examined that influenced homocysteine levels. CONCLUSIONS: Insulin resistance and hyperinsulinaemia in patients with PCOS is associated with elevated plasma homocysteine, regardless of body weight. This finding may have important implications in the short term regarding reproductive performance, and in the long term regarding cardiovascular complications associated with insulin-resistant PCOS.