Semen DNA fragmentation index, evaluated with both TUNEL and Comet assay, and the ICSI outcome.

BACKGROUND: In intracytoplasmic sperm injection (ICSI), there is always a risk of using spermatozoa with damaged DNA. The purpose of this study was to evaluate the percentage of spermatozoa with DNA fragmentation in processed semen samples used in ICSI cycles and to investigate the relationship between the DNA fragmentation index (DFI) and the ICSI outcome. PATIENTS AND METHODS: Fifty-six couples undergoing ICSI treatment were included. DFI was evaluated, by both terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and single cell gel electrophoresis (Comet) assays, in the processed semen samples used for ICSI. RESULTS: Of the processed semen samples 17.85% had > or =10% spermatozoa with fragmented DNA. There was no correlation between DFI and the ICSI outcome. DFI assessed by the TUNEL assay was negatively correlated with sperm concentration, progressive motility and sperm morphology. CONCLUSION: A considerable proportion of processed semen samples used for ICSI have a high DFI. However, DFI of the processed semen samples does not seem to be related to the ICSI outcome.

First pregnancy and life after preimplantation genetic diagnosis by polar body analysis for mucopolysaccharidosis type I.

Preimplantation genetic diagnosis (PGD) may help couples at risk to avoid pregnancies with known genetic diseases. In Germany, the only option to perform PGD is the analysis of polar bodies (PB). Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder. Q70X is one of the frequent diseases causing mutations of alpha-L-iduronidase (IDUA), leading to a severe phenotype with mental retardation and various somatic abnormalities, and making a request for PGD is understandable. Using five polymorphic DNA markers from the vicinity of IDUA, PGD on first PB was performed for a consanguineous couple, both heterozygotes of the Q70X mutation of IDUA. Sixteen first PB were obtained by laser assisted hatching of the zona pellucida. Genotyping led to the conclusion that 3/16 oocytes carried wild-type IDUA alleles. Only one of these oocytes showed pronucleus formation after intracytoplasmic sperm injection and was transferred on day 2 after oocyte retrieval. A singleton pregnancy was established. Prenatal diagnosis showed a fetus heterozygous for Q70X. For MPS I, PB analysis is a feasible way to perform PGD and it may be an acceptable alternative for couples with moral objections to embryo selection, or for countries in which genetic testing of the embryo is prohibited.

Factors affecting outcome after ICSI with spermatozoa retrieved from cryopreserved testicular tissue in non-obstructive azoospermia.

There is a lack of data regarding variables affecting the treatment outcome for non-obstructive azoospermia when spermatozoa from cryopreserved testicular specimens are utilized for ICSI. The objective of the present retrospective analysis was to investigate the effect of various parameters on treatment outcome in such cases. One hundred and sixty-five couples with non-obstructive azoospermic males undergoing a total of 297 cycles were included. In all cases the testicular tissue retrieved by multiple open-biopsy testicular sperm extraction was stored in liquid nitrogen and, after thawing, only mature spermatozoa were used for ICSI. When no motile spermatozoa were recovered, immotile spermatozoa were used. In 159 cycles, motile spermatozoa were utilized for ICSI, while in 138 cycles immotile spermatozoa were utilized. Higher normal fertilization rate (60.4 +/- 3.1 versus 51.3 +/- 1.6%, P < 0.05), number of embryos transferred (2.8 +/- 0.06 versus 2.6 +/- 0.04, P < 0.05), modified cumulative embryo score (31.2 +/- 1.6 versus 23.9 +/- 0.8, P < 0.001), and proportion of motile spermatozoa injected (67.8 versus 49.8%, P < 0.05) were observed in cycles that resulted in clinical pregnancies. Binary logistic regression analysis showed that sperm motility (odds ratio 2.06, 95% CI 1.1-3.9, P < 0.05), but not woman's age, number of treatment cycle, type of GnRH-analogue used for pituitary suppression, number of oocytes retrieved or number of embryos transferred was a significant determinant of the likelihood of clinical pregnancy. In conclusion, sperm motility after freeze/thawing of testicular tissue is the major determinant of the success of ICSI in non-obstructive azoospermia.

Cumulative pregnancy rate after ICSI with cryopreserved testicular tissue in non-obstructive azoospermia.

The aim of the present study was to describe a simplified and inexpensive method of testicular tissue freezing, to assess the cumulative clinical pregnancy rate (CPR) by this technique, and to provide useful information for counselling couples with non-obstructive azoospermia. One hundred and sixty-five couples with non-obstructive azoospermic males pursuing assisted conception, from December 1995 to December 2002, were included. In all cases, the testicular tissue retrieved by open multiple-biopsy (both sides, by testicular sperm extraction) was frozen using a simple liquid nitrogen vapour freezing technique and was stored in liquid nitrogen thereafter. Only mature spermatozoa were used for intracytoplasmic sperm injection (ICSI) after thawing. Expected CPR were calculated using the Kaplan-Meier survival analysis. A total of 281 cycles were performed resulting in 53 clinical pregnancies. Crude and expected CPR (95% confidence intervals) after three cycles were 32.1 (25.7-40.1) and 55.7% (37.0-74.4) respectively. In conclusion, this simplified method for freezing testicular tissue resulted in a satisfactory outcome after ICSI in cases of non-obstructive azoospermia.

Polar body diagnosis for hemophilia a using multiplex PCR for linked polymorphic markers.

Preimplantation genetic diagnosis (PGD) is usually performed on blastomeres. In Germany, the only possibility to perform PGD is by analysis of polar bodies. We performed PGD using polar bodies in a woman who is a carrier of hemophilia A. Multiplex PCR followed by nested fluorescent PCR for five linked polymorphic markers was established. From 11 analyzed polar bodies, only 1 showed alleles linked to the mutation. The corresponding oocyte was transferred and no pregnancy was established. As seen in other investigations, the rate of heterozygous first polar bodies is surprisingly high.

Lessons learned from introducing an in-vitro maturation programme into clinical practice.

The technology of in-vitro maturation (IVM) aims at maturing immature oocytes from the germinal vesicle (GV) stage to the metaphase 2 stage of the second meiotic division in vitro. Gametes are obtained by immature oocyte retrieval from small antral follicles (5-12 mm) present in polycystic ovarian syndrome patients or at the beginning of each cycle of normally menstruating woman. Maturation time in vitro takes about 28-36 h. After precise patient selection and adequate preparation, a high proportion of oocytes are able to resume meiosis in vitro. Intracytoplasmic sperm injection is the preferred method of fertilization. About 300 children have been born worldwide after IVM, and are reported to be healthy. Recent data suggests that IVM has the potential to replace standard ovarian stimulation in the near future. Nevertheless, several aspects of IVM regarding safety and efficacy remain unanswered and will have to be addressed. This paper reviews data concerning IVM in clinical practice and describesexperience from the establishment of an IVM programme that resulted in the first IVM pregnancy in Germany.

Unilateral follicular aspiration and in-vitro maturation before contralateral oocyte retrieval: a method to prevent ovarian hyperstimulation syndrome.

BACKGROUND: Five women undergoing intracytoplasmatic sperm injection (ICSI) considered to be at high risk of developing an ovarian hyperstimulation syndrome (OHSS) from March to July 2002 underwent unilateral follicular aspiration. METHODS: When >/=15 follicles of 12-15 mm diameter in each ovary and a serum estrodial level >/=2500 pg/ml was present, follicular aspiration was performed unilaterally without hCG administration. Ovarian stimulation was continued for 1-3 days in four women before human chorionic gonadotrophin (hCG) was given. In one woman hCG injection was administered at the evening after unilateral follicular aspiration. The oocyte retrieval from the contralateral ovary was performed 36 h after hCG injection. By unilateral follicular aspiration two to six germinal vesicle (GV) oocytes could be retrieved. After in-vitro maturation of those oocytes ICSI was performed. RESULTS: In four women one to two oocytes were fertilized and cryopreserved. In one case only one triploid pronucleus (3PN) was observed. At the contralateral ovum-pick up after hCG injection a median of 10 could be retrieved. After transfer of a median of 3 embryos, no pregnancy was achieved. Four of five patients developed a severe OHSS and were hospitalized for a median of 3 days. CONCLUSION: Unilateral follicular aspiration and continuation of stimulation therefore can not be recommended for the prevention of OHSS.

Comparison of cryopreservation outcome with gonadotropin-releasing hormone agonists or antagonists in the collecting cycle.

OBJECTIVE: To compare the pregnancy rates of frozen-thawed 2-pronucleate (2PN) oocytes obtained either in a long protocol or in an antagonist protocol and ovarian stimulation with either human menopausal gonadotropin (hMG) or recombinant follicular stimulating hormone (recFSH). DESIGN: Retrospective data analysis. SETTING: Academic infertility center. PATIENT(S): Three hundred forty-two infertile couples who underwent a transfer of cryopreserved 2PN oocytes. INTERVENTION(S): hMG (n = 194) or recFSH (n = 92) in a long protocol or hMG (n = 16) or recFSH (n = 40) stimulation under pituitary suppression with the GnRH antagonist Cetrotide was used. The 2PN oocytes were transferred after endometrial preparation using E(2) valerate and vaginal progesterone (Crinone 8% vaginal gel). MAIN OUTCOME MEASURE(S): Implantation, pregnancy, and abortion rates. RESULT(S): Implantation rates in the freeze-thaw cycles were 5.6% (hMG) and 3.8% (recFSH) with 2PN oocytes from the long protocol and 7% from the antagonist cycles, irrespective of whether hMG or recFSH was used. Pregnancy rates were similar independent of whether they resulted from the long-protocol cycles with hMG (15.4%) and recFSH (13.1%) or from the antagonist protocol cycles with hMG (25.0%) and recFSH (17.5%). CONCLUSION(S): The potential to implant is independent of the gonadotropin-releasing hormone analogue and gonadotropin chosen for the collection cycle when previously cryopreserved 2PN oocytes were replaced after thawing in the cleavage stage.