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The aim of this study was to examine the role of growth factors associated with angiogenesis and oxidative stress in the pathogenesis of spontaneous miscarriage.We performed a comparative mRNA expression analysis of VEGF, PlGF, Flt-1, Angiogenin and Endoglin using Real-Time PCR, in the placenta and decidua collected from 12 patients presenting with spontaneous abortion and from 14 women undergoing induced abortion, during the first and second trimester of pregnancy.The mRNA expression of Flt-1 was significantly upregulated in the placenta of spontaneous abortions (5.17-fold, IQR: 2.72-9.11,In this study, we observed higher levels of sFlt-1 mRNA expression in the placenta of spontaneous abortions, while expression of other growth factors in placenta and decidua remained constant. This suggests that an imbalance of sFlt-1 expression in the placenta might contribute to the pathogenesis of spontaneous abortion, probably

Many studies have shown that BRCA mutation is not only related to cancer but also to ovarian aging. Studies in both human and mice oocytes have shown that Double-strand breaks (DSBs) accumulate with age.A review of the literature was completed through the PubMed database aiming to find articles regarding BRCA 1,2 mutation and if they are related to early menopause in order to use them as predictive biomarkers in the near future. The research used keywords in numerous combinations, such as "BRCA 1,2 mutation," "menopause," "ovarian reserves," "AMH," "genome-wide association studies," and "biomarkers." The literature was limited in this specific topic. The initial research found 16 screened articles, 7 of which were not included because there were not relevant, as far as publications in non-English language.Genome-wide association studies (GWAS) have found 44 genetic loci that are related to variations when a female is about to have menopause. BRCA1 is involved in these 44 loci that are associated with the age of menopause. This review has gathered all results of literature search about the association between BRCA genes and early menopause. Most of the articles found that women with BRCA mutation have earlier menopause compared to non-carriers.In conclusion, in the near future BRCA1,2 genes could be used as predictive biomarkers of menopause.

Pharmacogenomics is a promising approach in the field of individualized medicine in in vitro fertilization (IVF) treatment that aims to develop optimized pharmacotherapy depending on the genetic background of each infertile woman, thus to ensure maximum effectiveness of the medication used, with minimal side effects. The unique genetic information of each infertile woman, in combination with already known, as well as new predictors of ovarian response and the progress of pharmacoepigenomics, is anticipated to greatly benefit the process of controlled ovarian stimulation. This review analyses current data on IVF pharmacogenomics, a new approach that is gradually moving to the frontline of modern IVF treatment.

Triple-negative breast cancer (TNBC) is characterized by a lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) and unfortunately is not associated with good prognosis. Treatment of breast cancer mainly depends on chemotherapy, due to the lack of specifically approved targeted therapies for TNBC. It is of paramount importance to find new therapeutic approaches, as resistance to chemotherapy frequently occurs. Herein, we present clinical studies published within the last five years, in order to reveal possible targeted therapies against TNBC. We aimed to discuss factors against TNBC, such as tyrosine kinase inhibitors, anti-androgens, poly ADP-ribose polymerase-1 (PARP-1) inhibitors, anti-angiogenic factors, immune checkpoints and histone deacetylase inhibitors (HDACI). Furthermore, the PI3K/AKT/mTOR pathway seems to be a promising field for the development of new anti-TNBC targeted therapies. Data from 18 clinical trials with patients suffering from TNBC were summarized and presented descriptively.

Tocolytic drugs are used widely in order to prevent preterm birth. Ritodrine, is the only food and drug administration (FDA) approved drug for tocolytic use. We estimated the cytogenetic effect of ritodrine administered as maternal therapy, alone or in combination with smoking, in women and their neonates.Lymphocyte and fibroblasts cultures were evaluated and three indices were analyzed; sister chromatid exchanges (SCEs), proliferation rate index (PRI) and mitotic index (MI) as well as average generation time (AGT) and population doubling time (PDT). Campothacin (CPT-11) was used as a positive control.Administration of ritodrine up to a month revealed significant reduction of SCEs/cell in neonates in the presence or absence of the mutagenic agent. A statistical significant increase on SCEs, for mothers and neonates, was noticed in neonate's lymphocytes when tocolytic therapy was over a month. Ritodrine revealed a cytoprotective action against smoking when the two factors were combined, but the synergistic action of ritodrine with smoking increased genotoxicity, cytostaticity and cytotoxicity of neonates after long administration (1-3 months).The time-depended genotoxic, cytostatic and cytotoxic action of ritodrine alone or in combination with smoking suggests that its administration should not exceed the time period of a month.

Adenosine is considered to be the brain's endogenous anticonvulsant as many studies have showed and it is responsible for seizure arrest and postictal refractoriness. Alterations in the adenosinergic system (adenosine and its receptors) have been referred by many previous studies indicating that deficiencies or modifications in the function of this purinergic system may contribute to epileptogenesis. Due to this emerging implication of adenosine in the managing of seizures, a new field of adenosine-based therapies has been introduced including adenosine itself, adenosine receptor agonists and antagonists and adenosine kinase inhibitors. The method with the least side effects (heart rate, blood pressure, temperature or even sedation) is being quested including intracerebral implantation of adenosine releasing cells or devices.

Although angiogenetic therapy using recombinant growth factors holds much hope for the treatment of ischaemic diseases, there are still many unanswered questions, including the method of administration, the correct dose of these factors, and the duration of the therapeutic approach. Exercise has also been suggested to induce neovascularizaiton in muscles. We evaluated the angiogenetic effects of the intramuscular administration of basic-fibroblast growth factor (b-FGF) and acidic-fibroblast growth factor (a-FGF) in rat heart, compared with rats who exercised daily. In conclusion, both the intramuscular administration of b-FGF and exercise increased significantly angiogenesis in the heart in contrast to the intramuscular administration of a-FGF, which was ineffective.

Angiogenic factors which control the angiogenic process represent a promising strategy for restoration of blood flow, but require further evaluation before clinical use. Exercise has also been reported to induce neovascularisation in muscles.To evaluate the angiogenic effects of basic fibroblast growth factor (b-FGF) and acidic fibroblast growth factor (a-FGF) on rat gastrocnemius muscle, when administered intramuscularly, and to compare them with those obtained by daily exercise.Forty nine rats were allotted to the following groups: A, controls; B, exercise by swimming; C1 and C2, intramuscular injection of b-FGF and a-FGF respectively; D1 and D2, b-FGF and a-FGF injection in combination with exercise. The antibody mouse anti-rat CD31 was used to evaluate the numbers of blood vessels present in histological preparations of gastrocnemius muscle.Significant increases in the numbers of blood vessels of the right gastrocnemius muscles in groups C1 and D1 were observed compared with controls (p0.05). There was only a slight increase in angiogenesis in the left gastrocnemius muscle of groups C1 and D1 compared with controls (p0.05), and there was a decrease in angiogenesis in the gastrocnemius muscle of the swimming group compared with controls.The intramuscular administration of b-FGF, but not a-FGF, induced significant local angiogenesis in gastrocnemius muscle at the site of injection.

The German embryo protection law (Embryonenschutzgesetz, ESchG) does not allow embryo selection, but only selection at the pronuclear stage. Furthermore, only as many pronuclear stage zygotes are allowed to be selected as are planned to be transferred in the same cycle. This means that after pre-selection of, for example, three pronucleated zygotes, these three must be transferred on the same or the subsequent day. A second selection process is not allowed. Non-selected pronuclear stage zygotes are allowed to be cryopreserved for a subsequent transfer. The same situation is present in other European countries such as Swizerland and Italy. it is illegal to cryopreserve an oocyte after fusion of the pronuclei (PN). The idea of these laws was to avoid ethical problems related to cryopreservation of surplus embryos or wastage of embryos, because these have, according to these laws, the status of individual persons. The current situation initiates much interest in developing a refined method of cryopreserving human pronuclear zygotes. The following article will discuss that issue in details.

Background. Women with a single ovary form a group of special interest in assisted reproduction. The aim of this study was to compare the outcome of intracytoplasmic sperm injection cycles between women with one and two ovaries. To our knowledge, this is the first study reporting the outcome of intracytoplasmic sperm injection cycles with women with a single ovary. Methods. Sixty-three intracytoplasmic sperm injection cycles in 24 women with a single ovary were compared with 191 intracytoplasmic sperm injection cycles in 109 women with both ovaries. All cycles were stimulated with triptorelin-long protocol. Results. Statistically significant differences were found in the following parameters between women with a single ovary and women with two ovaries: total number of administrated gonadotropins (63.22 ± 45.03 vs. 44.72 ± 21.92), number of follicles (8.29 ± 5.02 vs. 14.45 ± 7.94), estradiol peak levels (1695.05 ± 1177.34 vs. 2728.51 ± 1852.67), number of retrieved metaphase II oocytes (6.95 ± 3.78 vs. 11....

Objective: To investigate the ICSI outcome of the patients who produced four follicles or less after ovarian stimulation using frozen–thawed surgically obtained spermatozoa. Study design: The patient cohort of this study was composed of a carefully selected group of 20 men suffering from obstructive and non-obstructive azoospermia and in whom spermatozoa had been seen in their harvested epididymal aspirates and testicular tissues and the cryopreserved specimens had been used for subsequent ICSI cycles. This group of men represent those in whom the female partners produced only four follicles or less after ovarian stimulation. Results: For the cases of obstructive azoospermia with MESA ( n =6) a total of nine cycles was carried out. Four couples went through one cycle, one couple underwent two cycles, one couple accomplished three cycles. Out of the nine cycles, seven went to embryo transfer. No pregnancy occurred in the MESA–ICSI group of patients. The mean±S.D. number of oocytes per cycle was 2.556±1.236, the mean±S.D. number of embryos per transfer was 1.444±1.014. Two couples did not have an embryo replacement because of absence of fertilisation. The mean±S.D. number of gonadotropin ampoules was 53.88±37.30 and the mean±S.D. duration of ovarian stimulation was 13.38±4.534 days. For the cases of non-obstructive azoospermia with TESE ( n =14) a total of 16 cycles was carried out. Thirteen couples went through one cycle, one couple accomplished three cycles. Out of the 16 cycles, 11 cycles went to embryo transfer. One pregnancy occurred in the TESE–ICSI group of patients, which produced live offspring. The mean±S.D. number of oocytes per cycle was 3.00±1.211, the mean±S.D. number of embryos per transfer was 1.313±1.195. Five couples did not have an embryo replacement, four of them because of absence of fertilisation and one because of abnormal fertilisation (3 PN). The mean±S.D. number of gonadotropin ampoules was 81.77±53.40 and the mean±S.D. duration of ovarian stimulation was 16.71±3.667 days. Conclusion: In our opinion, these data show that it is possible to achieve satisfactory fertilisation rates using frozen–thawed epididymal and testicular spermatozoa obtained from men with obstructive or non-obstructive azoospermia, but the limiting factor in reaching the stage of embryo transfer is the poor ovarian response to stimulation induction. It therefore seems preferable to cancel these cycles, in the hope that a better response might be obtained in a subsequent cycle, avoiding in this way financial and emotional implications.

Objective: To report and analyse our experience with ICSI treatment in infertile women ≥40 years of age, with the intention of contributing to current debates on the effect of aging on the reproductive potential. Study design: 107 infertile couples in which the female partner was aged ≥40 years and who received ICSI treatment between January 1996 and December 1998. Results: A total of 107 women underwent 171 treatment cycles during this period. Of 171 cycles initiated 33 were cancelled (cancellation rate=19.3%). In this way, 17 women did not have embryo transfer at all, while 90 patients had 138 cycles with oocyte retrieval and successful embryo transfer, with a mean number of embryos per transfer 2.36. Sixteen pregnancies occurred and eight of them ended in spontaneous abortion. The implantation rate was 4.9%, the pregnancy rate per initiated cycle was 9.35% and per transfer cycle 11.59%. The miscarriage rate was 50%. Moreover, 12 patients had supernumerary embryos, that were cryopreserved and transferred in 17 thawing cycles and resulted in two pregnancies ending in abortion. All pregnancies occurred when three embryos were available, except in two cases with two available embryos. The great majority of the total pregnancies (16 of 18) resulting in women aged between 40 and 42 years. Conclusion: Our data show that women 40 and older with existing ovarian function may benefit from ICSI treatment, even when the indication for treatment is male factor infertility. Supernumerary embryos, that are cryopreserved and transferred in subsequent cycles can improve the overall pregnancy rates per oocyte retrieval, although these women should be aware of the very high risk of miscarriage.

Die intrazytoplasmatische Spermatozoeninjektion (ICSI) ist heute eine in der Routine etablierte Methode zur Behandlung schwerer Formen mannlich bedingter Subfertilitat. Neuere Untersuchungen haben ergeben, dass auch die Injektion unreifer Samenzellen, v. a. runder und elongierter Spermatiden, zur Fertilisierung und Teilung von Oozyten fuhren kann. Damit ergibt sich ein Ansatz, auch Kinderwunschpatienten mit postmeiotischem Reifungsarrest zu behandeln. Die Erfolge hinsichtlich ausgetragener Schwangerschaften reichen bis jetzt aber noch nicht an die durch Injektion von reifen Spermatozoen erzielten heran. Gerade bei der Injektion von Rundspermatiden ergeben sich fur das IVF-Labor Schwierigkeiten bei der Identifikation und der Unterscheidung von anderen runden Zellen und den fehlenden Vitalitatskennzeichen. Auserdem wird z. Z. diskutiert, ob durch die noch nicht vollendete Reifung von Gameten das Risiko genetischer Defekte erhoht wird. Die Injektion unreifer Spermatozoenvorstufen wird deshalb vorerst eine Methode bleiben, die nur in wenigen spezialisierten IVF-Zentren durchgefuhrt werden kann.

Treatment of severe male subfertility has become available since the intracytoplasmic injection of a single sperm into an oocyte was successfully applied for the first time in 1992. Moreover, also with the use of testicular spermatozoa for this procedure fertilization and pregnancies could be accomplished. This review addresses the development of these techniques and discusses achievements and problems as well as future aspects of the feasibility of early spermatid injection are stressed. Furthermore it includes the basic elements of spermatogenesis and the major concerns regarding the underlying genetic reasons for spermatogenic failure.

The objective of this retrospective clinical study was to assess the benefit of assisted fertilization in cases of anejaculatory infertility due to retrograde ejaculation. We report the outcome of intracytoplasmic sperm injection (ICSI) treatment. In 16 couples in which the men suffered from retrograde ejaculation. We performed 35 cycles of ICSI with spermatozoa retrieved from post-ejaculatory urine. The patients had been instructed to alkalinize the urine by ingesting sodium bicarbonate before the procedure. The fertilization rate averaged 51.2%. Seven clinical pregnancies were achieved. Three spontaneous first trimester abortions occurred, but three live offspring were delivered and one pregnancy is ongoing. In conclusion, the use of ICSI may be feasible for patients with retrograde ejaculation who are resistant to medical treatment and whose sperm quality is so low or unpredictable that intrauterine insemination or conventional methods of in-vitro fertilization are not possible.

The examination of the genotoxic, cytostatic and cytotoxic effects of smoking during pregnancy.Lymphocyte cultures of peripheral blood were received from 20 women who smoked during pregnancy as well as umbilical cord blood of their newborns. Fluorescence Plus Giemsa staining technique was used in order to perform cytogenetic analyses for three indices, Sister Chromatid Exchanges (SCEs), Proliferation Rate Index (PRI) and Mitotic Index (MI). To reveal any underlying chromosome instability, CPT-11 was used as a positive control.Newborns whose mothers smoke during pregnancy had increased SCEs levels on their lymphocytes when they were exposed to the mutagenic agent CPT-11 (p 0.01) compared with newborns lymphocytes exposed to the same agent with non-smoking mothers. Also, mothers smoking during pregnancy had increased SCE levels when their lymphocytes were exposed to CPT-11 (p 0.01) compared with non smoking mothers whose lymphocytes were exposed to the same agent. In both groups newborns appeared as having decreased (p 0.01) spontaneous SCEs levels compared with the corresponding SCE rates of their mothers. Decreases of PRIs and MIs are observed in mothers compared to their newborns.Smoking during pregnancy can promote cytogenetic damage in newborn's DNA, causing chromosome instability. The clinical importance of this indirect damage lies in the fact that this type of damage can act synergistically with other environmental and/or chemical mutagenic substances possibly leading to carcinogenicity.

To evaluate the latent and active forms of MMP-2 and MMP-9 in human semen samples and to investigate their association with semen parameters.Basic semen analysis was performed in 82 semen samples. Seminal plasma was analyzed with gelatin zymography.Both latent and active forms of MMP-2 and MMP-9 were detected in human seminal plasma. The latent forms were the predominant ones. MMP-2 and MMP-9, either in latent or active forms, were not correlated with semen parameters. ProMMP-9 levels were higher in semen samples with abnormally low concentration (or = 19 x 10(6)/ml) compared with semen samples with concentrationor = 50 x 10(6)/ml.MMP-2 and MMP-9 are both present in human semen. The latent forms of both MMPs are the predominant ones. ProMMP-9 is elevated in samples of low sperm concentration.

The intrafollicular levels of IGF-I and epidermal growth factor (EGF) were studied in women undergoing controlled ovarian hyperstimulation using the multidose GnRH-antagonist protocol or the long agonist protocol, in an attempt to elucidate whether GnRH-antagonists affect the levels of the two growth factors. The follicular fluid concentration of IGF-I, EGF, estradiol and progesterone were detected in 68 women undergoing ovarian hyperstimulation for intracytoplasmic sperm injection (ICSI) cycles. There were no differences in intrafollicular concentrations of EGF and IGF-I in the two studied groups. Additionally, we found no correlation between the intrafollicular levels of IGF-I or EGF and the ICSI outcome. The intrafollicular levels of IGF-I were positively correlated with those of progesterone. In conclusion, the intrafollicular levels of IGF-I and EGF do not seem to be influenced by the stimulation protocol. The intrafollicular levels of both growth factors can not serve as prognostic markers for the ICSI outcome.

Although angiogenic therapy using recombinant growth factors holds much hope for the treatment of ischaemic diseases, there are still many unanswered questions, including its effectiveness on atrophic muscles.To evaluate the angiogenic effects of intramuscularly administered basic fibroblast growth factor (b-FGF) on normal gastrocnemius muscles of rats and atrophic gastrocnemius muscles after tenotomy.Forty rats were divided into groups as follows: group A, controls; group B, injected with 1 microg b-FGF; group C, tenotomy performed on the right gastrocnemius muscle; group D, tenotomy and 1 microg b-FGF. Mouse anti-rat CD31 antibody was used to evaluate the number of blood vessels present in histological preparations.There was a significant (p0.01) decrease in the number of blood vessels compared with the controls in the atrophic muscles of group C. This was similar to the decrease in muscle weight in this group. However, there was a significant (p0.01) increase in the number of blood vessels compared with the controls in groups B and D. Similarly, there was a significant (p0.01) increase in the number of blood vessels in group D compared with the atrophic muscles in group C.Intramuscular administration of b-FGF increases angiogenesis in both normal and atrophic rat gastrocnemius muscles at the injection area.

The advent of recombinant gonadotrophins brought significant changes in fertility therapy. Treatment options with recombinant gonadotrophins add more to knowledge on folliculogenesis and ovarian steroidogenesis. Over a decade, recombinant LH (rLH) has been used for clinical trials, and the amount of peripheral LH that is necessary for optimal follicular growth, oocyte maturation, subsequent embryo development and assisted reproduction outcome during ovulation induction can now be better evaluated. This review evaluates the effect of rLH supplementation on ovarian stimulation and assisted reproduction outcome. The studies conducted with rLH supplementation in ovarian stimulation in different groups of patients and in cases of controlled ovarian stimulation are clearly discussed in this review.

The outcome of freeze-thaw cycles with pronuclear stage oocytes or embryos, derived from collecting cycles stimulated with gonadotropin-releasing hormone (GnRH)-antagonists’ protocols, was reviewed. The viability of cryopreserved pronuclear stage oocytes and embryos, the quality of transferred embryos and the pregnancy rates of the freeze-thaw cycles seem to be satisfactory regardless of the type and dose of GnRH-antagonist.

Sperm donation seems to be the only solution in cases of negative results with testicular sperm extraction (TESE). However, it implies a genetic dissociation between husband and offspring that results in psychological stress, as well as ethical and existential dilemmas for the couple. Facing these dilemmas, some couples prefer father-to-son donation as an alternative solution. However, father-to-son sperm donation involves many potential medical, ethical and emotional risks. A broad discussion within the scientific community on these issues should contribute towards a better approach of these cases.

The present retrospective study evaluated the outcome of frozen-thaw cycles with oocytes obtained either during a multiple dose protocol of cetrorelix, or after the use of a gonadotrophin-releasing hormone (GnRH) agonist. A total of 101 subfertile couples were included. These couples had a total of 222 transfers of frozen-thawed pronuclear oocytes after IVF/intracytoplasmic sperm injection (ICSI) treatment. According to the stimulation protocol during various cycles, four groups were established: cetrorelix/recombinant FSH (recFSH) (69 cycles), cetrorelix/human menopausal gonadotrophin (HMG) (10 cycles), GnRH-agonist/recFSH (71 cycles) and GnRH-agonist/HMG (72 cycles). The transfer cycles were mildly stimulated with transdermal oestradiol. No statistically significant difference was seen among the four groups regarding post-thaw survival rate, cumulative embryo score, implantation rate and pregnancies. Frozen-thawed pronuclear oocytes obtained with the use of cetrorelix give satisfactory implantation and pregnancy rates, similar to those obtained with a GnRH-agonist. These results do not depend on the gonadotrophins (HMG or recFSH) used in the collecting cycle.

Objective: To estimate the efficacy of gonadotropin-releasing hormone (GnRH) antagonist ‘Cetrorelix’ in poor responders comparing with the standard long protocol. Design: The study population consisted of 21 poor responders who underwent ICSI and treated with Cetrorelix according to the multiple-dose protocol and who were compared with 21 poor responders treated according to the long protocol and who also underwent ICSI. Patients in both groups were matched for chronological age, the number of follicles found by ultrasound at the retrieval day and cause of infertility. Fifteen patients of GnRH antagonist group were treated with the combination of GnRH antagonist with clomiphene citrate (CC) plus gonadotropins, while six patients were treated with the combination of GnRH antagonist plus gonadotropins, but without CC. Results: The use of GnRH antagonist in a multiple dose protocol gave a pregnancy rate of 14.28% which was in the range expected for patient with poor response, but with shorter treatment duration and with fewer ampoules of gonadotropins as compared with the use of a GnRH agonist protocol in a depot formulation. Within Cetrorelix group patients who received CC had a significant shorter duration of stimulation and needed fewer ampoules as compared with patients in the same group who did not receive CC. Conclusions: A GnRH antagonist multiple dose protocol may be the protocol of choice for the treatment of poor responders. The use of GnRH antagonist Cetrorelix ended with significantly less ampoules of gonadotropins and a shorter duration of stimulation.

This retrospective study was performed to examine the implantation and pregnancy rates of frozen–thawed pronuclear stage oocytes obtained with the use of a GnRH antagonist, Cetrorelix (Cetrotide® ASTA-Medica, Frankfurt/M, Germany) used in a multidose protocol with hMG, and to compare these results with those obtained after a conventional long GnRH analogue protocol (Decapeptyl-Depot, Ferring, Kiel, Germany). The study population consisted of 31 infertile couples with frozen–thawed pronuclear stage oocytes after ICSI treatment using the GnRH antagonist Cetrorelix (Cetrorelix®) and 31 infertile couples with frozen–thawed pronuclear stage oocytes after ICSI treatment using the long GnRH analogue protocol. Patients underwent ICSI after down regulation with a GnRH agonist (Decapeptyl) and stimulation with hMG, or a GnRH antagonist (Cetrorelix) and hMG. The supernumerary pronuclear stage oocytes were cryopreserved and transferred in a later mildly stimulated cycle. The implantation and pregnancy rates for frozen–thawed pronuclear stage oocytes derived from the GnRH antagonist compared with the GnRH agonist were 3.26% versus 3.73% (P=1.0000) and 8.33% versus 10.25% (P=1.0000), respectively. To our knowledge we report here the first pregnancies obtained by the transfer of cryopreserved pronuclear stage embryos generated from ICSI using a GnRH antagonist in the collecting cycle. The use of Cetrorelix in a multiple dose protocol in combination with hMG does not demonstrate a negative effect on viability, implantation potential or pregnancy outcome as compared to 2PN conceptuses obtained from a long GnRH agonist-hMG protocol.

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.