F. Nodar

Birth of twin males with normal karyotype after intracytoplasmic sperm injection with use of testicular spermatozoa from a nonmosaic patient with Klinefelter’s syndrome

OBJECTIVE To report the birth of healthy twin males after the use of testicular spermatozoa from a nonmosaic patient with Klinefelters syndrome. DESIGN Case report. SETTING Private reproduction center with university affiliation. PATIENT(S) A couple undergoing intracytoplasmic sperm injection (ICSI) combined with testicular sperm extraction because of the husbands secretory azoospermia and a nonmosaic 47,XXY peripheral blood karyotype. The wife, a healthy female, presented with a history of oligomenorrhea. INTERVENTION(S) ICSI was performed using testicular spermatozoa; 3 mM pentoxifylline solution was used to induce sperm motility because the spermatozoa recovered were all immotile. MAIN OUTCOME MEASURE(S) Normal fertilization, embryo cleavage, pregnancy outcome, and peripheral blood karyotype of the newborns. RESULT(S) Thirteen metaphase II oocytes were injected. Seven of them fertilized normally and six did not fertilize. Three good-quality embryos (4-cell stage class II) were transferred, and four were cryopreserved at the two-cell and four-cell stages using a slow freezing protocol. Twelve days after ET, a beta-hCG determination was positive. Ultrasonographic examination revealed three intrauterine fetal sacs, but one of them showed a fetal pole without cardiac activity and vanished in subsequent ultrasonographic examinations. The patient delivered twins with normal male peripheral blood karyotypes. CONCLUSION(S) Normal outcome after the use of testicular sperm extraction and ICSI in a nonmosaic patient with Klinefelters syndrome reaffirms the notion of low transmission risk of this gonosomal aneuploidy.

Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia.

OBJECTIVE To establish the predictive value of serum inhibin B levels as an indicator of the presence of testicular spermatozoa in nonobstructive azoospermia, compared with the traditional serum FSH marker. DESIGN Prospective study. SETTING Private high-complexity reproductive center with university affiliation. PATIENT(S) Seventy-eight patients with nonobstructive azoospermia, 15 patients with obstructive azoospermia, and 10 fertile volunteers. INTERVENTION(S) Blood samples, testicular sperm extraction, percutaneous epididymal sperm aspiration, and semen collection. MAIN OUTCOME MEASURE(S) Serum levels of inhibin B and FSH and presence of spermatozoa on TESE, PESA, or regular semen analysis. RESULT(S) Patients with nonobstructive azoospermia has significantly higher levels of serum FSH and significantly lower levels of inhibin B. Mean inhibin B serum levels were significantly higher in patients with nonobstructive azoospermia who had spermatozoa on TESE than in those in whom no spermatozoa were found (89.31 +/- 73.24 pg/mL vs. 19.23 +/- 22.34 pg/mL), but mean FSH serum levels did not have similar predictive power (21.37 +/- 12.92 IU/mL vs. 19.27 +/- 10.28 IU/mL). The cut-off level of inhibin B separating both groups, as determined by the receiver-operating characteristic curves, was >53 pg/mL. CONCLUSION(S) Serum inhibin B level seems to be more accurate than serum FSH level in prediction of the presence of testicular spermatozoa in patients with nonobstructive azoospermia.

Healthy baby born after reduction of sperm DNA fragmentation using cell sorting before ICSI.

Magnetic activated cell sorting (MACS) with annexin V microbeads recognizes externalized phosphatidylserine (PS) residues on the surface of apoptotic spermatozoa. The successful use of this novel technique applied to a highly apoptotic semen sample before performing intracytoplasmic sperm injection (ICSI) is reported here. The use of annexin V microbeads for selecting non-apoptotic spermatozoa seems to reduce the percentage of altered cells, improving the chance of pregnancy after ICSI.

Microtubules and parental genome organisation during abnormal fertilisation in humans

We analysed the distribution of beta-tubulins, acetylated alpha-tubulins and chromatin configuration in 113 human zygotes showing abnormal fertilisation, 16-18 h after conventional in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). After a first characterisation using phase contrast microscopy, immunofluorescence staining was performed in 67 IVF and 46 ICSI zygotes that developed one, three or more pronuclei and/or subnuclei, with or without extrusion of the second polar body. Independently of the number of pronuclei found, beta-tubulins were uniformly distributed throughout the cytoplasm of the abnormal zygotes. We did not observe any kind of microtubule alteration with respect of the ploidy level and/or its origin. The most frequent abnormal fertilisation pattern found after IVF was the presence of three or four pronuclei (74.6%). On the other hand, the presence of one pronucleus (63.0%) was the main pattern found after ICSI. No differences between the two groups were seen in terms of development of subnuclei. Anamolies detected after IVF and ICSI showed different aetiologies such as parthenogenetic activation, gynogenetic or androgenetic development, as well as digynic or diandric fertilisation.

Pronuclear abnormalities and cytoskeletal organization during assisted fertilization in a patient with multifollicular ovarian response.

AbstractPurpose: To analyze the distribution of α tubulins and acetylated α tubulins and the chromatin configuration in abnormally fertilized zygotes from a patient with a multifollicular ovarian response after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Methods: Immunofluorescence and phase contrast microscopy was performed in abnormally fertilized zygotes. Results: After phase contrast microscopy analysis, immunofluorescence staining was performed in 20 oocytes that developed ≥3 pronuclei (PN) and karyomeres after IVF–ICSI. Around 80% of the abnormal zygotes from IVF were the consequence of monospermic fertilizations. Retention of the second polar body (PB) and the presumptive split of ≥1 PN within the cytoplasm were the main events present in most oocytes after IVF–ICSI. Conclusions: Fluorescence labeling of selected sperm and oocyte components affords a unique view of abnormal fertilized zygotes. Surprisingly, anomalies detected after IVF–ICSI showed similar etiologies in this special group of zygotes.

Relationship Between Sperm DNA Fragmentation and Nuclear Vacuoles.

OBJECTIVE The aim of the present study is to assess the correlation between the presence, quantity and size of nuclear vacuoles and DNA damage and chromatin status in sperm samples of men who underwent to assisted reproduction technology. METHODS Forty six males who underwent to assisted reproductive technology (ART) were considered. According to their latest semen analysis (<3 months), were grouped into: (A) strict morphology index ≤4% (26) and (B) strict morphology index ≥14% (20). Motile sperm were selected by density gradient, and MSOME study was conducted to assess the number and size of nuclear vacuoles. DNA fragmentation (TUNEL) and DNA strand status (acridine orange) were assessed over the selected spermatozoa accordingly to their vacuole pattern. RESULTS In group A, sperm without vacuoles (1°) have similar levels of DNA fragmentation (TUNEL) in compare to the rest of observed patterns (2°- 6°). Regarding to AO, spermatozoa with large or several vacuoles that cover more than 30-50% of the nuclear surface are AO+, but not necessarily TUNEL positive. The first three patterns of vacuoles patterns had lower levels of AO in compare to grades 4° and 6°. In group B, those sperm with one or more vacuoles greater than 30%-50% (4° and 6°), had a significant increase in TUNEL values, in relation to group 1°- 3°. Considering AO, it was found that the 4° and 6° pattern had a significantly elevated level of this marker, as same of group A (P <0.05). CONCLUSIONS There is no relationship between the greater number and size of sperm vacuoles with high levels of DNA fragmentation in patients with severe teratozoospermia (Kruger <4%). Conversely, this relationship is evident in normal semen samples (normal morphology. Sperm selection by IMSI technique, to select non-fragmented sperm in patients with Kruger <4%, is not necessarily secured when non-vacuolated sperm is selected.

Correlation between Cytoplamic Oocyte Maturation and Chromosomal Aneuploidies - Impact on fertilization, embryo quality and pregnancy.

OBJECTIVE To establish the relationship between oocyte cytoplasmic maturation and its chromosomal status and determine the effect of this feature over the reproductive outcome in patients with sub-optimal fertilization in ART. METHODS Fifty couples who underwent ART were selected. From nineteen patients, 22 metaphase II-MII and 18 failed-fertilized oocytes after ICSI were studied. The first polar body was collected for chromosomal analysis by aCGH. Oocytes were processed by immunocytochemistry (ICC) to determine oocyte maturation: assessment of inactive MPF status and the conformation-alignment of the metaphase plate.Other 31 couples presented sub-optimal fertilization (<50%) after ICSI, and failed-fertilized oocytes were studied by ICC. Two groups were conformed according to the main feature observed: A) cytoplasmic immaturity and sperm premature chromosome condensation and B) sperm nuclear decondensation failure with mature cytoplasm. RESULTS Regarding MII mature oocytes, 87% had a normal metaphase plate and 84% were chromosomally normal. Contrary, immature oocytes presented abnormal metaphase plate (86%) and just 33% were euploid. In failed-fertilized oocytes: 100% of mature oocytes had a normal metaphase plate and 71% were euploid. When oocytes were cytoplasmic immature, 37% of them were normal (metaphase plate) and 50% were chromosomally normal.The global rate of aneuploidies and metaphase plate disarrangements in immature oocytes (MII+failed-fertilized) were significantly higher than mature oocytes (P<0.05).In patients with sub-optimal fertilization, the percentage of top quality embryos and pregnancy rate was significantly higher in group B (P<0.05). CONCLUSION Oocyte cytoplasmic immaturity is related to metaphase plate anomalies and aneuploidies. Fertilized oocytes, from a cohort with sub optimal fertilization with cytoplasmic immaturity, had poorer reproductive outcomes.

Simultaneous expression analysis of deleted in azoospermia-family genes and CDC25A: their potential as a predictor for successful testicular sperm extraction.

infections, varicocele, hypogonadotropic hypogonadism, chromosome abnormalities, and obstruction or agenesia of the seminal ducts. The study was designed in accordance with the Helsinki Declaration and its last modification (Tokyo 2004) on human experimentation, and it was approved by the Ethics Committees from Universidad Maimónides and the Centro de Estudios en Genética y Reproducción. Informed Consent was obtained from all patients. The diagnosis of azoospermia was established on the basis of the independent analysis of, at least, two semen samples collected 1 week apart. The serum concentrations of FSH (normal range: 1.5–7 mIU ml−1), LH (normal range: 1.1–9 mIU ml−1), and testosterone (normal range: 10–30 pmol ml−1) were measured and fell into the normal range in all patients. Azoospermic patients underwent a diagnostic testicular biopsy and sperm retrieval (TESE) by microsurgery and agreed to provide a small piece of testicular tissue (5 mm in diameter) for research purposes. The testicular histopathology was categorized according to the most advanced degree of spermatogenesis, and the biopsies were classified either as HS (n = 5) or MA (n = 3), according to McLachlan et al. (2007).10 DAB (3,3’-diaminobenzidine) immunohistochemistry was performed to localize DAZ family proteins and CDC25A in each biopsy. Negative controls were processed simultaneously by omitting the primary antibody or preabsorbing the primary antibody with specific synthetic peptides. Relative quantitation of gene expression by Real-time PCR of the DAZ gene family and CDC25A was calculated using standard curves and normalized to actin in each sample. Immunohistochemical analysis showed that in all biopsies with HS and MA, expression of DAZ was mainly detectable in the cytoplasm of spermatogonia clusters, near the basal lamina of the seminiferous tubules (Figure 1a), and DAZL was detectable in the cytoplasm of some spermatogonia and spermatocytes (Figure 1b). BOULE and its downstream substrate CDC25A shared a similar pattern of expression in germ cell cytoplasm (Figure 1c and 1d). Interestingly, in one biopsy diagnosed with MA, no immunoexpression of both proteins was detected, but we were able to detect BOULE mRNA in this particular biopsy, suggesting that the translation of BOULE might be regulated by another RNA-binding protein. We did not found an association between the expression levels of the DAZ family genes analyzed by real-time PCR with the diagnosis of Dear Editor, Infertility is a major health problem affecting 10%–15% of couples seeking to have children, and a male factor can be identified in about half of these cases.1 Nonobstructive azoospermia is one of the causes of male infertility (10%), resulting from testicular failure.2 The most common histological patterns in these patients are hypospermatogenesis (HS), maturation arrest (MA), and Sertoli cell-only syndrome (SCO).3 The search for sensitive and specific markers of spermatogenesis that could better predict the sperm retrieval rates in patients with nonobstructive azoospermia can lead to improved management of male infertility. DAZ (Deleted in Azoospermia) gene family has been extensively studied because the microdeletions containing DAZ genes in the Y chromosome are associated with a variety of testicular failures and impaired spermatogenesis.4–6 DAZ gene family consists of two autosomal genes, BOULE and DAZ-L (DAZ-like), and the DAZ gene cluster on chromosome Y. These genetic factors encode for RNA-binding proteins that are mainly expressed in germ cells and are considered essential for male fertility.5 Recently, the members of the cell cycle regulators CDC25 family were recognized as potential substrates for DAZ family proteins. Particularly, CDC25A is abundantly expressed in the testis and functions in the G1-S transition and M-phase exit, suggesting a role in mitotic or meiotic regulation of spermatogenesis.7–9 The analysis of single DAZ gene has shown that its dysfunction leads to abnormal spermatogenesis and may cause infertility. They were, however, poorly associated to sperm recovery during assisted reproductive treatments. Whether the simultaneous expression of the members of the DAZ gene family and its substrates may provide better information of testicular damage and success of sperm recovery in infertile patients has not been yet analyzed. We evaluated eight men (29–38-year-old) with idiopathic infertility and nonobstructive azoospermia diagnosed by open testicular biopsy. None of these patients showed genitourinary Simultaneous expression analysis of deleted in azoospermia‐family genes and CDC25A: their potential as a predictor for successful testicular sperm extraction