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We have reported recently the first birth after intrauterine transfer of embryos obtained by injection of round spermatids into oocytes in cases of unexpected azoospermia. Here we provide a complete documentation of the series of 11 cases in which this novel method of infertility treatment was employed. In four of these cases, elongated spermatids were identified in the ejaculate, and it was decided to perform elongated spermatid injection (ELSI). In the other six cases, only round spermatids were present, and round spermatid injection (ROSI) was done. In one case, ROSI was given preference to ELSI because of a very poor viability status of elongated spermatids present in the ejaculate. Fertilization of at least one oocyte was achieved in 10 of the 11 treatment cycles; the fertilization rate in these 10 cycles ranged between 7 and 100% with a mean value of 45%. All of the two-pronucleated zygotes cleaved and were transferred to the patient’s uterus. A singleton pregnancy was achieved in two ROSI cycles. Both pregnancies developed uneventfully and resulted in the birth of normal infants. These data show that intra-ooplasmic injection of spermatids obtained from the ejaculate may become the treatment of first choice in patients with non-obstructive azoospermia. [ABSTRACT FROM PUBLISHER]

Human oocytes that failed to display signs of fertilization by 44 h after intracytoplasmic sperm injection (ICSI) were processed for electron microscopic analysis. All oocytes were arrested at metaphase II. The first polar body contained intact cortical granules and chromosome clumps, which were not surrounded by a nuclear envelope but still associated with microtubules. When a second globular body was present, it always showed the same ultrastructure, indicating that it had originated from fragmentation of the first polar body and not from the resumption of the second meiotic division. The most prominent organelles of the oocyte cytoplasm were the smooth endoplasmic reticulum and mitochondria. In the oocyte cortex, cortical granules were intact, with no signs of incipient or incomplete cortical reaction. Oocyte chromosomes were found in the oocyte periphery near the locality of the first polar body extrusion. They consisted of dense aggregates of chromatin associated with microtubules. The chromatin of the injected spermatozoon was demembranated and partially decondensed. In some cases, vesicular and tubular structures, apparently of oocyte origin, were associated with the periphery of the sperm chromatin mass but they never formed a continuous layer. These data suggest that fertilization failure after ICSI is basically a failure of oocyte activation. [ABSTRACT FROM AUTHOR]

This study was undertaken to assess the potential of aged human oocytes from failed in-vitro fertilization attempts as a model for the study of fertilization events after subzonal sperm insertion (SUZI). Criteria of aged oocyte suitability for this purpose were (i) the absence of nuclei, (ii) the presence of a polar body, (iii) the absence of cell division or fragmentation, (iv) marked ooplasmic contraction in hyperosmotic medium, and (v) rapid ooplasmic relaxation after returning into normo-osmotic medium following the micromanipulation. Micro-injection techniques were essentially the same as for SUZI with fresh oocytes. Oocytes that fused with the micro-injected spermatozoa developed pronuclei of typical internal structure. However, the number of pronuclei was often higher than that theoretically expected if each sperm nucleus incorporated into the oocyte gave rise to a single pronucleus. Thus, the use of aged oocytes implies the need for a specific method to assess the frequency of fusion in sperm samples examined. The results suggest that the method described here can be applied in a preliminary diagnostic test before a therapeutic SUZI attempt and in studies aimed at the optimization of sperm treatment protocols to increase the fusion capacity of subzonally inserted spermatozoa. [ABSTRACT FROM AUTHOR]

Preliminary evidence has suggested that the phosphodiesterase inhibitor pentoxifylline augments the fertilizing potential of asthenozoospermic sperm samples, presumably by improving sperm movement. Here, we used computer-assisted sperm movement analysis to compare the effects of pentoxifylline in normozoospermic and asthenozoospermic specimens. The study focused on the following issues: the changes in individual movement characteristics in response to pentoxifylline, the rapidity of the response, the effect of sperm capacitation on the response, the persistence of the response after drug removal and the variability of responses among asthenozoospermic individuals. Data obtained show that (i) pentoxifylline increases the curvilinear velocity, path velocity, straight-line velocity, lateral head displacement, beat cross frequency and sperm hyperactivation in both normozoospermic and asthenozoospermic specimens, (ii) pentoxifylline does not modify the percentage of motile spermatozoa, (iii) the pentoxifylline effect reaches a maximum within 10 min of treatment in fresh semen as well as in capacitated sperm suspensions and persists for at least 2 h after drug removal and (iv) pentoxifylline improves the movement characteristics in most asthenozoospermic individuals. Results are discussed with regard to methods of therapeutic application of pentoxifylline as an enhancer of sperm movement in assisted reproductive technology. [ABSTRACT FROM AUTHOR]

The nature of oocyte cytoplasmic factors controlling the development of the male pronucleus was investigated by inseminating human, zona-free oocytes at metaphase of the 1st and 2nd meiotic division. Oocytes at metaphase of the 2nd meiotic division could support the full structural and functional development of male pronuclei, whereas the vast majority of those at metaphase of the 1st meiotic division failed to do so. This suggests that oocyte cytoplasmic factors required for male pronuclear formation do not develop fully until the oocyte reaches the 2nd meiotic metaphase. When increasing numbers of spermatozoa entered one oocyte, the transformation of sperm nuclei into pronuclei was impaired progressively. The later stages of pronuclear development were particularly sensitive to polyspermy. These factors should be taken into consideration in the development of techniques of micromanipulation-assisted insemination. [ABSTRACT FROM PUBLISHER]

The present era of infertility management is characterized by an increasing interest in defects of sperm-egg interactions. When the spermatozoon meets the egg, it must first react with the enveloping coat which controls sperm access to the cell surface of the oocyte. Adequate sperm motility is a major condition for penetration through the egg investments. The ability of spermatozoa to respond to physiological stimuli by a timely acrosome reaction is another necessary prerequisite for this event, as well as for sperm fusion with the oocyte. The clinical significance of current laboratory tests evaluating different aspects of gamete function is discussed and a practical guide is given for their indication and interpretation and for the use of their results in choosing rational therapy. [ABSTRACT FROM PUBLISHER]

Focuses on sperm-induced calcium oscillations. Relationship between calcium oscillations and cell growth and differentiation; Identification and cloning of high molecular weight protein; Sperm-oocyte fusions.

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STUDY QUESTION Does the health status of infants fathered by nonmosaic Klinefelter syndrome (KS) patients whose partners underwent ICSI with sperm obtained from testicular dissection reveal any genetic risk for the offspring?. SUMMARY ANSWER KS patients undergoing testicular sperm extraction (TESE) are capable of conceiving healthy children. WHAT IS KNOWN ALREADY Paternity has been successfully achieved in nonmosaic KS patients (47,XXY karyotype) by ICSI using either ejaculated or testicular spermatozoa. A crucial concern is the potential transmission of genetic abnormalities to the offspring. Some studies reported that 47,XXY spermatogonia are capable of completing spermatogenesis leading to the production of mature spermatozoa with increased aneuploidies. Other authors showed that where focal spermatogenesis is present in nonmosaic KS males, it originates from euploid germ cells and, therefore, produces normal mature gametes. In support of this finding, at present, the great majority of children born from nonmosaic KS patients are chromosomally normal. STUDY DESIGN, SIZE, DURATION From April 2004 to June 2010, 38 azoospermic patients with nonmosaic KS were examined for the presence of testicular spermatozoa. Spermatozoa were retrieved from 15 patients and 26 ICSI cycles were done (16 with cryopreserved sperm). There were 15 pregnancies leading to the birth of 16 babies who were karyotyped at amniocentesis and after birth. PARTICIPANTS/MATERIALS, SETTING, METHODS Participants were recruited from couples attending the European Hospital, Rome, and Clinica MAR&Gen, Granada, for infertility treatment. Both the European Hospital and Clinica MAR&Gen are private clinics. Testicular tissue was extracted with TESE or micro-TESE. After retrieval, fresh sperm was used for ICSI or it was cryopreserved for future use. MAIN RESULTS AND THE ROLE OF CHANCE Spermatozoa were retrieved from 15 patients (14 TESE and 1 micro-TESE) out of 38 (39.5%). A total of 26 ICSI cycles were performed: 10 with fresh and 16 with cryopreserved-thawed sperm. Mean ages (y) of patients with positive and negative sperm retrieval were, respectively, 34.8 ± 1.72 and 35.6 ± 4.08 (NS, nonsignificant). Comparing ICSI cycles performed with fresh sperm (n = 10) to those performed with frozen–thawed sperm (n = 16): Fertilization rates per injected oocyte were 53.0% (44 of 83) and 47.8% (32 of 67), respectively (NS). The cleavage rate per injected oocyte was 90.6% (29 of 32) versus 68.2% (30 of 44); P = 0.026. Clinical outcomes were not significantly different between the fresh and the frozen–thawed sperm group: clinical pregnancy rates were 7 of 10 (70.0%) and 8 of 16 (50.0%); implanted embryos (per transferred embryo) were 8 of 23 (34.8%) and 8 of 29 (27.6%); delivery rates were 6 of 10 (60.0%) and 5 of 16 (31.3%). Sixteen babies were born, all of them are healthy with a normal karyotype, eight from the fresh sperm group and eight from the frozen–thawed sperm group. LIMITATIONS, REASONS FOR CAUTIONS The small numbers available for study mean that only common problems can be excluded. WIDER IMPLICATIONS OF THE FINDINGS This study provides further reassurance that KS men can father healthy children and that pre-implantation genetic diagnosis on embryos conceived with their sperm is not strongly indicated. However, until conclusive information is available, such couples should be offered extensive genetic counseling. STUDY FUNDING/COMPETING INTEREST(S) No external funding was obtained for the present study. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER Not applicable. [ABSTRACT FROM PUBLISHER]

Italian legislation regarding reproductive medicine limits the number of embryos transferred per attempt to three. Thus, in order to achieve pregnancy, more IVF cycles may be required, generating a need for methods of ovarian stimulation with fewer side effects. The gonadotrophin-releasing hormone (GnRH) antagonists have several advantages in this respect, but there is a debate regarding a possible lower pregnancy rate from resulting cycles. This study evaluated the clinical applicability of GnRH antagonists for ovarian stimulation in young women undergoing intracytoplasmic sperm injection (ICSI) in which only three oocytes can be fertilized. The 200 women treated with GnRH antagonist had a significantly shorter stimulation and lower gonadotrophin consumption, oestradiol concentration, total and mature oocyte recovery as compared with 200 matched controls treated with GnRH agonist. No differences were found between the groups in the number of normal zygotes, total cleaved, transferred and high quality embryos, or in the clinical outcomes. Thus, the previously reported lower pregnancy rate in GnRH antagonist cycles may be related to the oocyte characteristics. Finally, under conditions of oocyte number restriction, the GnRH antagonist-based cycles may be proposed as an efficacious, safe and minimally invasive alternative to GnRH agonist in a standard long protocol. [ABSTRACT FROM AUTHOR]

Recombinant FSH (r-FSH) used for ovarian stimulation can currently be self-administered either by a conventional syringe or by a pen device. This randomized controlled trial compares the efficacy and convenience of a new more sophisticated and fully automated injection device (Softinject™) with the conventional syringe for r-FSH self-administration. A total of 300 women needing ovarian stimulation for IVF/intracytoplasmic sperm injection were randomized to the automated injector or the conventional syringe group. Patients of both groups had ovarian stimulation with follitropin a after pituitary desensitization with a gonadotrophin-releasing hormone agonist. State anxiety score, overall pain score and pregnancy rate were chosen as the main outcome measures. Patients in the automated injector group showed lower state anxiety (P <0.01) and overall pain (P<0.01) scores and a comparable pregnancy rate per started cycle as compared with the conventional syringe group. They needed lower doses of r-FSH (P<0.05) and their stimulation was shorter (P< 0.05). It is concluded that the use of a fully automated injector for r-FSH self-administration reduces pain and stress as compared with the conventional syringe. This device can be used for any subcutaneously administered drug employed in ovarian stimulation. [ABSTRACT FROM AUTHOR]

BACKGROUND: The clinical efficacy of the current methods used for cryopreservation of metaphase II human oocytes is low. Meiotic spindle disorders are thought to be largely responsible for this situation. METHODS: Supernumerary fresh metaphase II human oocytes were cryopreserved in 1,2‐propanediol with 0.1 M sucrose using a slow freezing/rapid thawing programme. Meiotic spindles were analysed in these living metaphase II oocytes at sequential steps of the freezing and thawing procedures with the use of a computer‐assisted polarization microscopy system (Polscope). RESULTS: The meiotic spindle was detected in all 56 oocytes (from 16 patients) before freezing and remained visible in all these oocytes throughout the preparation for freezing up to the time that they were loaded into cryopreservation straws. Immediately after thawing, the spindle was visible in 35.7% of oocytes, but it disappeared in all of the thawed oocytes during the subsequent washing steps. However, the spindle reappeared in all surviving thawed oocytes after washing (57.4%), by 3 h of incubation at 37°C in culture medium. CONCLUSIONS: The current techniques of oocyte freezing and thawing inevitably cause meiotic spindle destruction. All spindles observed in thawed oocytes result from post‐thaw reconstruction. [ABSTRACT FROM AUTHOR]

The X-linked dominant form of Charcot--Marie--Tooth syndrome (CMTX) is a clinically and genetically heterogeneous hereditary disorder of the peripheral nerves caused by mutations in the GJB1 gene that encodes a gap junction protein named connexin 32 (Cx32). Clinically, CMTX is characterized by peripheral motor and sensory deficit with muscle atrophy. A couple with a previous history of pregnancy termination after being diagnosed positive for CMTX by chorionic villus sampling, was referred for preimplantation genetic diagnosis (PGD). The female partner carried the causative H94Q, characterized by a C→G substitution in codon 94 of exon 2 of the GJBI gene. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the mother's mutation using polymerase chain reaction (PCR), followed by mutation analysis performed using the minisequencing method. Amelogenin sequences on the X and Y chromosomes were also co-amplified to provide a correlation between embryo gender and mutation presence. A single PGD cycle was performed, involving nine fertilized oocytes, five of which developed into good quality embryos useful for biopsy. Two unaffected embryos were transferred, resulting in a singleton pregnancy followed by the birth of a healthy female. [ABSTRACT FROM AUTHOR]