First live birth in China after cryopreserved ovarian tissue transplantation to prevent premature ovarian insufficiency.

OBJECTIVE: This article reports the first live birth after cryopreserved ovarian tissue transplantation to prevent premature ovarian insufficiency in China. METHODS: A patient with myelodysplastic syndrome received ovarian tissue cryopreservation before hematopoietic stem cell transplantation, and six ovarian cortex strips were thawed and transplanted into her peritoneal pocket 2 years later. RESULTS: Pregnancy occurred spontaneously 27 months after grafting, and a healthy girl was born at 38 weeks gestation. Until now, the child has developed normally without any major diseases. CONCLUSIONS: We report the first live birth resulting from ovarian tissue cryopreservation and transplantation in China.

First pregnancy in China after ovarian tissue transplantation to prevent premature ovarian insufficiency.

OBJECTIVE: This article reports the first case of pregnancy after frozen-thawed ovarian tissue transplantation to prevent iatrogenic premature ovarian insufficiency in China. METHODS: Ovarian tissue cryopreservation was performed in a patient with myelodysplastic syndrome (MDS) before multi-agent chemotherapy and hematopoietic stem cell transplantation. Two years later, she showed complete remission from MDS, and six frozen-thawed ovarian tissue strips were transplanted into the peritoneal pocket. RESULTS: The patient's ovarian activity was restored 3 months after transplantation, and pregnancy occurred spontaneously 27 months after grafting. Until now, the pregnancy has progressed for 30 weeks, and the repeated ultrasound showed normal fetal development. CONCLUSION: This is the first pregnancy resulting from ovarian tissue cryopreservation and transplantation in China.

Ovarian tissue cryopreservation for patients with premature ovary insufficiency caused by cancer treatment: optimal protocol.

Objective: Premature ovary insufficiency is frequent after chemotherapy/radiotherapy in cancer patients. Ovarian tissue (OT) cryopreservation and later retransplantation, the routine method in Europe, has recently been implemented at the first center in China. We investigated the protective effect of the antioxidant N-acetyl-l-cysteine (NAC) during the decisive freezing-thawing steps. Methods: Fifteen OT samples were obtained from each of 13 cancer patients prospectively and randomly assigned to a control group and four groups with different NAC concentrations (Group 1, 0 mM NAC; Group 2, 0.5 mM NAC; Group 3, 1 mM NAC; Group 4, 5 mM NAC; Group 5, 25 mM NAC). After thawing, the follicle viability, DNA fragmentation, levels of reactive oxygen species (ROS), and total antioxidant capacity (TAC) were evaluated. Results: OT cryopreserved and thawed with 25 mM NAC (Group 5) has the lowest proportion of apoptotic stroma cells, but the worst follicle viability. The other four groups show similar anti-apoptosis and good follicle viability. Group 4 presented the lowest ROS and highest TAC levels. Conclusions: OT cryopreserved and thawed in medium supplemented with 5 mM NAC shows the highest antioxidant and lowest ROS capability, good apoptotic parameters, and follicle viability. Our results need to be confirmed in larger patient cohorts prior to being accepted as a standard protocol.

Artificial oocyte activation: evidence for clinical readiness.

Artificial oocyte activation using Ca(2+)ionophores or similar compounds is a widely applied technique in IVF laboratories. This is all the more interesting as most of the agents aiming for intracellular Ca(2+) increase do not result in physiological Ca(2+) oscillations but much rather cause a single Ca(2+) transient. Two observations from mammals may explain why a rather non-physiological single Ca(2+) peak caused by ionophores is sufficient to rescue cycles showing severe male factor infertility, deficient oocyte maturation, developmental problems in humans, or both. On the one hand, it has been shown that it is mainly the initial Ca(2+) rise that drives further downstream events, in particular calcium/calmodulin-dependent protein kinase II (CaMKII) action, and on the other, it is possible that this enzyme remains active even in the absence of Ca(2+). It therefore seems that mammalian oocytes can respond to a wide range of intracellular Ca(2+) signals and have a surprisingly high degree of tolerance for changes in cytosolic Ca(2+). As epigenetic consequences or differences in gene expression have not been studied to date, artificial oocyte activation has to be considered as experimental and should only be applied with a proper indication.

Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

Efficacy of ovarian tissue cryopreservation in a major European center.

PURPOSE: To evaluate the effect of cryopreservation and thawing of ovarian tissue from oncological patients opting for fertility preservation on ovarian tissue viability. METHODS: In this prospective cohort study, the ovarian tissue viability before and after cryopreservation and thawing was measured for 25 newly diagnosed oncological patients who had their ovarian tissue cryopreserved. Outcome measures were follicle integrity (histology), follicle viability (Calcein viability assay), steroid hormone production (estradiol and progesterone production in vitro) and overall tissue viability (glucose uptake in vitro). This study was conducted at a Cryobank for storage of ovarian tissue in a university hospital. RESULTS: Cryopreserved/thawed ovarian tissue showed a decreased glucose uptake when compared to tissue that had not been cryopreserved. In addition, a diminished E2 and P4 production was observed after cryopreservation and thawing, despite the fact that numbers of viable follicles as determined by the Calcein viability assay were comparable. Histological examination revealed a higher percentage of degenerated follicles after cryopreservation and thawing. CONCLUSIONS: Ovarian tissue cryopreservation and thawing impairs the viability of ovarian tissue in oncological patients opting for fertility preservation.

[Preimplantation diagnosis--PID: preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)]. / Präimplantationsdiagnostik - PID : Präimplantative genetische Diagnostik (PGD) und präimplantatives genetisches Screening (PGS).

Preimplantation diagnosis (PID) comprises all the relevant diagnostic procedures for the investigation of genetic, structural, or numerical changes of the genetic information in spermatozoa and oocytes as well as in embryos after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). PID of oocytes is well established in Germany for the above-mentioned indications. PID at the embryonic level, i.e., trophectoderm biopsy of blastocysts, is possible in centers with proven expertise in reproductive medicine and human genetics. A high risk for genetic disease in the child or a high likelihood for stillbirth or miscarriage is a prerequisite for PID. A specialized ethics committee is required to look into each case before making a decision. While PID is still under development in Germany, it has been a well-established technology worldwide for 24 years. International experience in PID and the resulting implications are discussed in this article.

Temperature-induced sperm nuclear vacuolisation is dependent on sperm preparation.

The influence of temperature during incubation on the degree of sperm nuclear vacuolisation was assessed by two different experiments. In a first experiment, motile spermatozoa from 24 patients were prepared by the swim-up technique and incubated either at room temperature or at 37 °C for up to 4 h. The presence of sperm nuclear vacuoles was determined by contrast-enhanced high magnification microscopy. No statistically significant difference was found in the degree of sperm nuclear vacuoles in both groups (RT: 45.6 ± 17.6%; 37 °C: 48.4 ± 17.0%) following 4 h of incubation. In a second experiment, spermatozoa from six patients were either prepared by swim-up or washed and incubated at 37 °C. After 4 h of incubation, a significant increase in sperm nuclear vacuolisation was found in washed sperm (from 51.5 ± 15.4% to 68.6 ± 9.0%; P < 0.05) but not in swim-up sperm (from 51.5 ± 15.4% to 48.2 ± 17.1%; n.s.). Our data show that the mode of sperm preparation does influence sperm nuclear vacuolisation at 37 °C (Experiment II). However, sperm nuclear vacuolisation is unaffected by temperature in motile sperm after preparation and isolation by swim-up.

ESHRE PGD Consortium/Embryology Special Interest Group--best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS).

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium published a set of Guidelines for Best Practice to give information, support and guidance to potential, existing and fledgling PGD programmes (Thornhill AR, De Die-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN et al. ESHRE PGD Consortium best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2005;20:35-48.). The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of ESHRE's recent advice on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme; Organization of a PGD centre, fluorescence in situ hybridization-based testing, amplification-based testing and polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Here we have updated the sections that pertain to embryology (including cryopreservation) and biopsy of embryos prior to PGD or PGS. Topics covered in this guideline include uses of embryo biopsy, laboratory issues relating to biopsy, timing of biopsy, biopsy procedure and cryopreserving biopsied embryos.

Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential.

It has previously been demonstrated that zona pellucida imaging of human oocytes using polarized light microscopy is a clinically applicable method for the noninvasive assessment of oocyte quality. This study was designed to investigate whether zona pellucida characteristics of bovine oocytes and zygotes in polarized light may similarly serve as a useful marker for developmental competence in bovine reproductive biotechnologies. Zona birefringence intensity parameters of 2862 oocytes/zygotes were objectively evaluated with an automatic analysis system and correlated with oocyte/zygote quality. In detail, immature oocytes of good quality assessed with brilliant cresyl blue staining showed significantly lower zona birefringence than poor-quality counterparts (P<0.001). After in vitro maturation and classification according to maturational status, the birefringence intensity parameters were significantly different in those oocytes that reached metaphase II compared with arrested stages (P<0.001). Following either parthenogenetic activation or IVF with subsequent in vitro culture in a well-of-the-well system until day 9, superior development as determined by cleavage, blastocyst formation, and hatching ability was associated with lower zona birefringence intensity parameters. When early zygote-stage embryos were selected and assorted in groups based on zona birefringence (high/medium/low), the group of embryos derived from high-birefringence zygotes displayed a significantly compromised developmental potential compared with low-birefringence zygotes. These results clearly show that developmentally competent bovine oocytes/zygotes exhibit lower zona birefringence intensity parameters. Therefore, birefringence imaging of zona pellucida is a suitable technique to predict bovine preimplantation embryo development.

Polar body biopsy: a viable alternative to preimplantation genetic diagnosis and screening.

Polar body diagnosis (PBD) is a diagnostic method for the indirect genetic analysis of oocytes. Polar bodies are by-products of the meiotic cell cycle, which have no influence on further embryo development. The biopsy of polar bodies can be accomplished either by zona drilling or laser drilling within a very short time period. However, the paternal contribution to the genetic constitution of the developing embryo cannot be diagnosed by PBD. The major application of PBD is the detection of maternally derived chromosomal aneuploidies and translocations in oocytes. For these indications, PBD may offer a viable alternative to blastomere biopsy as the embryo's integrity remains unaffected, in contrast to preimplantation genetic diagnosis (PGD) by blastomere biopsy. The rapid pace of developments in the field of molecular diagnostics will also influence the advantages of PBD, and probably allow more general diagnostic applications in the future.

Regional hippocampal atrophy in multiple sclerosis.

Gray matter brain structures, including deep nuclei and the cerebral cortex, are affected significantly and early in the course of multiple sclerosis and these changes may not be directly related to demyelinating white matter lesions. The hippocampus is an archicortical structure that is critical for memory functions and is especially sensitive to multiple insults including inflammation. We used high-resolution MR imaging at 3.0 T to measure hippocampal volumes in relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients and controls. We found that both groups of MS patients had hippocampal atrophy and that this volume loss was in excess of global brain atrophy. Subregional analysis revealed selective volume loss in the cornu ammonis (CA) 1 region of the hippocampus in RRMS with further worsening of CA1 loss and extension into other CA regions in SPMS. Hippocampal atrophy was not correlated with T2-lesion volumes, and right and left hippocampi were affected equally. Volume loss in the hippocampus and subregions was correlated with worsening performance on word-list learning, a task requiring memory encoding, but not with performance on the Paced Auditory Serial Addition Task (PASAT), a test of information processing speed. Our findings provide evidence for selective and progressive hippocampal atrophy in MS localized initially to the CA1 subregion that is associated with deficits in memory encoding and retrieval. The underlying histopathological substrate for this selective, symmetric and disproportionate regional hippocampal vulnerability remains speculative at this time. Further understanding of this process could provide targets for therapeutic interventions including neuroprotective treatments.

Symposium: innovative techniques in human embryo viability assessment. Oocyte assessment and embryo viability prediction: birefringence imaging.

Embryo viability is a key element for success in assisted reproduction. Since the beginning of the era of assisted reproduction treatment, embryo viability was mostly considered to be a function of developmental progression during the preimplantation phase. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems in assisted reproduction has enabled the detection of structures within oocytes that possess a natural birefringence. Birefringence imaging of the meiotic spindle and the zona pellucida in living animal and human oocytes represents a new approach in the assessment of oocyte and embryo viability. The technique was applied in several studies to select oocytes in order to improve treatment success. This review will summarize the present knowledge of birefringence imaging. The various applications in basic and clinical research as well as in clinical treatment will be presented, especially with regard to their effect on assisted reproduction.

Simplified technique of human ovarian tissue freezing: quick cooling from -36 degree C.

Standard protocol of freezing of human ovarian tissue presupposes the very slow cooling (-0.3 C/min) from auto-seeding to -40 C, then slow cooling (-10 C/min) to -140 C and then direct plunging into liquid nitrogen. The aim of this investigation was to compare the -10 C/min cooling rate of human ovarian tissue from -40 C to -140 C with the -220 C/min cooling rate (direct plunging into liquid nitrogen) from -36 degree C. After post-thawing in vitro culture of tissue, hormonal activity as well as follicle viability was evaluated. After culture of fresh tissue pieces (Group 1), pieces after freezing and thawing with slow cooling (-10 C/min) from -40 C (Group 2) and pieces after freezing and thawing with direct plunging into liquid nitrogen (-220 C/min) from -36 C (Group 3), the supernatants showed estradiol 17-ss concentrations of 481, 441 and 459 pg per ml, respectively, and progesterone concentrations of 9.05, 5.06, 4.87 ng per ml, respectively. It is concluded that 94, 96, and 98 percent follicles for Groups 1, 2 and 3, respectively, were normal. Technique of human ovarian tissue cryopreservation with very slow cooling to -36 C and then direct plunging into liquid nitrogen with -220 C/min cooling rate is tolerated without apparent detriment.

Presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane.

We investigated the presence and localization of oscillin in human spermatozoa in relation to the integrity of the sperm membrane, which was assessed by the hypo-osmotic swelling (HOS) test. We found no gross differences in the presence of oscillin in semen samples from men who presented with 70%, 40%, 25% or 2% of membrane-intact spermatozoa. By immunofluorescence, membrane-intact (HOS-positive) spermatozoa showed staining of a single band at the equatorial region, whereas over 80% of HOS-negative spermatozoa consistently showed a diffuse distribution of oscillin over the sperm head. However, some individuals presented with up to 50% of HOS-positive spermatozoa showing an aberrant localization of oscillin. We found a significant correlation rate (r=0.70, P < 0.05) between the percentage of HOS-positive spermatozoa with an equatorial oscillin localization and the fertilization rates achieved after intracytoplasmic sperm injection. These data suggest that the localization of oscillin in human spermatozoa might have an impact on egg activation and fertilization rates.

Characterization of testicular mouse glucosamine 6-phosphate deaminase (GNPDA).

Mammalian glucosamine 6-phosphate deaminase (GNPDA) was first detected in hamster spermatozoa. To further elucidate its role, we have cloned mouse GNPDA and produced a polyclonal rabbit anti-GNPDA antibody. This antibody recognized a 33 kDa protein in soluble extracts from mouse brain, liver, kidney, muscle, ovary, testis and sperm. Immunofluorescent analysis of the localization of GNPDA in male reproductive tissue revealed its presence in spermatids and in spermatozoa. In spermatids, GNPDA localized close to the developing acrosome vesicle and in spermatozoa close to the acrosomal region. Following the induction of the acrosome reaction, GNPDA fluorescence in spermatozoa was either reduced or GNPDA was absent. These data suggest that GNPDA might play a role in the acrosome reaction.

Activation of mouse oocytes requires multiple sperm factors but not sperm PLCgamma1.

A sperm cytosolic factor is responsible for oocyte activation at fertilization in mammals. The molecular identity of this factor is not yet known, although a sperm phospholipase Cgamma (PLCgamma) is a potential candidate. In this study, cation-exchange chromatography with a Heparin column was used for the fractionation of porcine sperm cytosolic extracts. Oocyte activation potential of the resulting fractions was tested and active fractions were subjected to Western blot analysis using antibodies specific to PLCgamma1. PLCgamma1 was detected in fractions other than those supporting oocyte activation (Ca(2+)-release and pronuclear formation). The active Heparin fraction was then purified on a Mono Q anion-exchange column. One of the resulting fractions still contained Ca(2+)-releasing activity, but pronuclear formation did not occur. We conclude that sperm PLCgamma1 is not involved in oocyte activation and that Ca(2+)-release and pronuclear formation requires multiple factors from sperm cytosol.

Laser-assisted microdissection of the zona pellucida facilitates polar body biopsy.

OBJECTIVE: To investigate whether polar body biopsy can be performed after laser microdissection of the zona pellucida (ZP). DESIGN: Mouse zygotes were allocated randomly to three groups. The zygotes were subjected to laser microdissection of the ZP and polar body biopsy (group 1), laser microdissection alone (group 2), or no treatment (group 3). SETTING: University-based IVF program. PATIENT(S): Animal study. INTERVENTION(S): A hole was drilled in the ZP of mouse zygotes using a 1.48-micron noncontact diode laser. A microneedle was inserted and the polar body was aspirated. MAIN OUTCOME MEASURE(S): The efficacy of polar body biopsy after laser microdissection of the ZP was evaluated. RESULT(S): The laser diode beam allowed for precise drilling of a 14- to 18-micron hole in the ZP. Polar bodies could be aspirated without damaging the zygote and did not disintegrate during the biopsy. Zygotes developed to blastocysts and underwent the same hatching as control zygotes. Lower hatching rates were observed in untreated zygotes. CONCLUSION(S): Laser microdissection of the ZP with a noncontact laser system facilitates subsequent polar body biopsy. The use of blunt-ended micropipettes greatly reduces the risk of damage to the zygote or the polar body. This procedure makes polar body biopsy more accurate and effective for preimplantation genetic diagnosis.

Cytogenetic and andrological status and ICSI-results in couples with severe male factor infertility.

AIM: To pursue whether cytogenetic aberrations correlate with specific spermatological or hormonal abnormalities. METHODS: 305 infertile couples were investigated. All male partners were referred to a complete andrological work-up with physical examination, determination of hormones, HIV testing and semen analysis. Cytogenetic analysis was carried out in both partners by means of standard techniques using cultured lymphocytes from peripheral blood. RESULTS: Among the 305 couples, 10 men (3.2%) and 10 women (3.2%) showed constitutional chromosomal aberrations, including reciprocal translocations (n = 7), Robertsonian translocations (n = 3), inversions (n = 3), other structural aberrations (n = 4) and sex chromosome aberration (n = 3). In addition to the impaired sperm count in most of the patients, a tendency to an increased proportion of spermatozoa with acrosome defect was observed. CONCLUSION: Chromosomal aberrations may contribute to the low fertilization and pregnancy rates in the infertile couples.

Stimulation of bone growth through sports. A radiologic investigation of the upper extremities in professional tennis players.

This contribution addresses the following questions: Does unilateral sports-specific strain affect the skeletal system of the athlete? Specifically, can any differences be found in longitudinal growth of the bones of the forearm and hand in professional tennis players between the stroke arm and the contralateral arm? An investigation was conducted involving 20 high-ranking professional tennis players (12 male and eight female players) between 13 and 26 years of age as well as 12 controls of the same age range. The radiologic examinations of the bones of the forearm and hand yielded an increase in density of bone substance and bone diameter as well as length in the stroke arm as compared with the contralateral arm. Whereas the first results confirm previous findings, the stimulation of longitudinal growth has never been reported. This change in bone structure and size can be attributed to two factors: mechanical stimulation and hyperemia of the constantly strained extremity. It may thus be regarded as a biopositive adaptation process.