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Polycystic Ovarian Syndrome (PCOS) affects 5-20 % of females and is the most common cause of anovulatory infertility. Leptin seems to have an important role in reproduction. Many reproductive pathologies such as preeclampsia, PCOS, and endometriosis are associated to plasma adiponectin levels. Kisspeptin levels are increased in PCOS women.A review of the literature was completed through the PubMed database aiming to find articles regarding leptin, adiponectin and kisspeptin and if they are related to PCOS pathogenesis.Even today it is not clear what is the role of leptin in women with PCOS, although most of the researchers found increased levels of leptin as well as leptin resistance in PCOS (both obese and lean individuals). Many more longitudinal studies should be done to vdiscover the usefulness of measuring adiponectin in prepubertal women who apparently have a possibility to develop PCOS to find out if they finally develop PCOS. Most of the researchers found that PCOS women have decreased levels of adiponectin unrelated to BMI levels. Nevertheless, not all studies had the same result. Moreover, it is necessary more studies to be made to investigate the connection between kisspeptin and other metabolic factors such as LH and insulin resistance.In general, it remains inconclusive whether leptin, adiponectin, and kisspeptin can be used as clinical and/or biochemical markers of PCOS. Therefore, it is essential to review the current data with regards to the association between PCOS and circulating leptin, adiponectin, and kisspeptin in women with PCOS.

Miscarriage is one of the most common complications of pregnancy. Although chromosomal abnormalities of the embryo is a well-known cause of miscarriage, a lot of cases remain unexplained, with immunologic and vascular growth alterations being considered as probable causes. Chemokines are produced by a variety of cells and exhibit several functions including both pro and anti-angiogenic properties. In this study, we investigated the role of the angiogenic and angiostatic chemokines in placenta and decidua tissues from spontaneous and induced abortions.Total RNA was extracted from the placenta and decidua tissues, which was then purified and converted into cDNA. Real-time PCR was then performed for the expression of the angiogenic CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 and CXCL4, and the angiostatic CXCL9, CXCL10, CXCL11, CXCL12 and CXCL14 and results were then statistically analyzed.Regarding the placenta, CXCL7 (2.29-fold, 2.16-2.38, p 0.05), CXCL4 (1.01-fold, 0.74-4.447, p 0.05), CXCL9 (0.87-fold, 0.43-1.34, p 0.05) and CXCL11 (0.31-fold, 0.22-0.45, p 0.05) were altered in spontaneous abortions. CCL2, CCL5, CXCL2-3, CXCL8, CXCL10, CXCL12 and CXCL14 were not statistically significant altered. Regarding the decidua, CXCL7 (7.13-fold, 6.32-7.54, p 0.01), CXCL8 (11.02-fold, 8.58-13.45, p 0.05), CCL20 (1.21-fold, 0.29-1.89, p 0.05) and CXCL9 (5.49-fold, 3.67-6.39, p 0.05) were overexpressed in spontaneous abortions. CXCL2-4, CCL2, CCL5, CXCL10-12 and CXCL14 did not show any differences. The expression of the chemokines CXCL1, CXCL5-6 was absent in either tissue or group.Our results show that the overexpression of angiostatic and diminished expression of angiogenic chemokines takes place in the placenta and decidua of spontaneous abortions, suggesting that dysregulation of angiogenesis could be a contributive factor to the pathogenesis of miscarriage.

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.

Triple-negative breast cancer (TNBC) is an extremely diverse group of breast tumors, with aggressive clinical behavior, higher rates of distant recurrence and worse overall survival compared to other types of breast cancers. The genetic, transcriptional histological and clinical heterogeneity of this disease has been an obstacle in the progression of targeted therapeutic approaches, as a ubiquitous TNBC marker has not yet been discerned. In terms of that, current studies focus on the classification of TNBC tumors in subgroups with similar characteristics in order to develop a treatment specialized for each group of patients. To date, a series of gene expression profiles analysis in order to identify the different molecular subtypes have been used. Complementary DNA microarrays, PAM50 assays, DNA and RNA sequencing as well as immunohistochemical analysis are some of the methods utilized to classify TNBC tumors. In 2012, the Cancer Genome Atlas (TCGA) Research Network conducted a major analysis of breast cancers using six different platforms, the genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays, in order to assort the tumors in homogenous subgroups. Since then, an increasing number of breast cancer data sets are being examined in an attempt to distinguish the classification with biological interpretation and clinical implementation. In this review, the progress in molecular subtyping of TNBC is discussed, providing a brief insight in novel TNBC biomarkers and therapeutic strategies.

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.

The use of open carriers for embryo vitrification has raised safety concerns and therefore vitrification in closed systems has been proposed. However, the drop in the cooling rate emerges as a major drawback. The objective of the present study was to compare the efficiency of vitrification in open versus closed conditions. Blastocysts were randomly allocated either to open ultra-rapid vitrification (group I) or closed aseptic vitrification (group II). In group I, blastocysts were exposed to two solutions of ethylene glycol/dimethylsulphoxide (10%/10% and 20%/20%), while in group II, blastocysts were pretreated with a solution of lower concentration (5%/5%). A total of 208 and 224 vitrification-warming cycles were performed for groups I and II, respectively. Both groups were equal in terms of maternal age, sperm parameters and number and quality of blastocysts vitrified, warmed and transferred per cycle. Importantly, there was no significant difference between the groups in the analysed outcomes; embryo survival rate (84.1% versus 82.1%), clinical pregnancy rate (45.9% versus 42.4%), implantation rate (25.6% versus 24.5%), cycle cancellation rate (6.7% versus 8.5%) and live birth rate (41.2% versus 41.0%). These data suggest that ultra-rapid vitrification may be replaced by aseptic vitrification without affecting clinical efficiency.

Background The use of immature oocytes derived from stimulated cycles could be of great importance, particularly for urgent fertility preservation cases. The current study aimed to determine whether in vitro maturation (IVM) was more successful before or after vitrification of these oocytes. Materials and Methods This prospective study was performed in a private in vitro fertilization (IVF) center. We collected 318 germinal vesicle (GV) oocytes from 104 stimulated oocyte donation cycles. Oocytes were divided into two groups according to whether vitrification was applied at the GV stage (group 1) or in vitro matured to the metaphase II (MII) stage and then vitrified (group 2). In the control group (group 3), oocytes were in vitro matured without vitrification. In all three groups, we assessed survival rate after warming, maturation rate, and MII-spindle/chromosome configurations. The chi-square test was used to compare rates between the three groups. Statistical significance was defined at P

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.

BACKGROUND The aim of our study was to explore cytokine and hormonal profiles in blood and follicular fluids from normal women stimulated with either the multidose antagonist or the long agonist protocol. METHODS Fifty-six patients were stimulated with the multidose antagonist protocol and 12 with the long agonist protocol. Interleukin (IL)-1beta, IL-6, tumour necrosis factor-alpha (TNFalpha), leptin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), estradiol (E(2)), progesterone and testosterone levels were measured in serum and follicular fluids by immunoassays. RESULTS The two treatment groups had similar cytokine concentrations in serum. The intrafollicular concentrations of IL-1beta, IL-6, VEGF and leptin were also similar in the two groups. The concentrations of bFGF in follicular fluids from the antagonist group (169.5 +/- 113.2 ng/ml) were lower than those from the agonist group (249.7 +/- 119.8 ng/ml). bFGF concentrations were correlated with the amount of administered gonadotrophins (R = 0.364, P < 0.01) which was significantly lower in the antagonist group (antagonist group: 2037.7 +/- 725.8 IU; agonist group: 2836.4 +/- 1163.5 IU). CONCLUSIONS Normal women stimulated with either the multidose antagonist or the long agonist protocol generally have similar cytokine profiles in serum and follicular fluids. The intrafollicular levels of bFGF tend to be lower in antagonist cycles because of the lower amount of administered gonadotrophins.

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.

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.

Chromosomal abnormalities are thought to be responsible for implantation failure, and among chromosomal abnormalities in normally developing embryos, aneuploidy is the most frequent. Genetic testing of preimplantation embryos for chromosomal aneuploidy allows selection of chromosomally normal embryos, and early detection of chromosomal aberration will increase the chance of conceiving. Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS), performed by polar body or blastomere analysis, is used in infertile patients treated with assisted reproduction technologies, especially in those with a poor prognosis, e.g. repeated IVF failure, advanced maternal age, or recurrent spontaneous abortion. The aim of this paper is to clarify the impact of PGD-AS in repeated implantation failure. In this review, the data collected so far regarding PGD-AS in this patient group will be discussed in depth.

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....

To investigate possible associations between serum anti-Müllerian hormone (AMH) levels, the number of oocytes collected and the availability and suitability of produced embryos for cryopreservation in in-vitro fertilization (IVF) cycles.Sixty women in their first IVF/intracytoplasmic sperm injection (ICSI) cycle were studied. The short stimulation protocol was used for controlled ovarian hyperstimulation. AMH levels were measured during the menstrual cycle preceding treatment.A strong, positive correlation between AMH and the number of collected oocytes was found. The patients with available and suitable embryos for cryopreservation had significantly higher levels of AMH.AMH appears to be a valuable marker mainly for ovarian reserve and response to IVF treatment. AMH levels are strongly associated with the number of retrieved oocytes and the availability of supernumerary embryos suitable for cryopreservation.

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.

Binding of FAS ligand (FASL) to its physiological receptor FAS, induces the activation of caspase-8, which triggers cell death. The FAS-FASL system regulates germ cell death. In this study, the role of the FAS-FASL system in male infertility was examined.72 samples were used (age=38.76 ± 9.06 years). Basic semen analysis was performed according to the WHO Laboratory Manual. Soluble (s) forms of FAS and FASL were measured in seminal plasma using commercially available immunoassay kits.Among the examined samples, 24 were normal and 48 abnormal, as evaluated by basic semen analysis. sFAS and sFASL levels in abnormal samples were slightly higher than in the normal ones. In all samples, sFAS correlated negatively with pH. In normal samples, sFAS was positively correlated with sperm concentration. In abnormal samples, sFAS strongly correlated with sFASL.Both factors of the FAS system were detected in seminal plasma. Further studies are necessary to shed light into the possible role of FAS-FASL system in male infertility.

We investigated the effects of the gonadotropin-releasing hormone (GnRH) agonist triptorelin as well the GnRH antagonist cetrorelix those of on the viability and steroidogenesis in human granulosa luteinized (hGL) cell cultures.The hGL cells were obtained from 34 women undergoing ovarian stimulation for IVF treatment. The cells were cultured for 48 h with or without 1 nM or 3 nM of cetrorelix or triptorelin in serum-free media. The cell viability was evaluated by the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. The concentrations of estradiol and progesterone in culture supernatants were measured by ELISA.Treatment with triptorelin slightly increased cell viability, whereas treatment with 3 nM cetrorelix led to a significant decrease. Estradiol concentrations were reduced with 3 nM triptorelin. Cultures treated with high-dose of either cetrorelix or triptorelin tended to secrete less progesterone than controls.Cetrorelix significantly reduces the viability of hGL cells. Triptorelin and cetrorelix may have minor effects on steroidogenesis. These results suggest that GnRH analogues may influence ovarian functions.

Objective To report on the capability of vitrified immature human oocytes to undergo in vitro maturation, fertilization, and embryo development, and to report on the post-thaw survival of vitrified embryos. Design Case report. Setting University-based IVF unit. Patient(s) Two women (34 and 36 years old) undergoing IVF therapy. Intervention(s) Immature oocytes, retrieved after ovarian stimulation, were vitrified. Post-thaw, they underwent in vitro maturation and fertilization by intracytoplasmic sperm injection (ICSI). The produced embryos were vitrified at day 2. In one case, the embryos were thawed and transferred. Main Outcome Measure(s) Post-thaw survival of vitrified immature oocytes, capability for in vitro maturation, fertilization, embryo development; post-thaw survival of vitrified embryos. Result(s) In the first case, six immature oocytes survived after thawing and matured after in vitro maturation. Five of them underwent ICSI, four fertilized, and three cleaved. The embryos were vitrified for future use. In the second case, two out of three immature oocytes survived after thawing. They were normally fertilized and cleaved, and the embryos were vitrified. Later, the embryos were thawed and transferred to the patient. Conclusion(s) These cases demonstrate that vitrified immature oocytes can undergo post-thaw in vitro maturation and fertilization. The produced embryos are capable to undergo vitrification and thawing.

The aim of this study was to measure circulating and intrafollicular concentrations of three inflammatory cytokines from women undergoing ovarian stimulation in order to determine their prognostic value in the outcome of intracytoplasmic sperm injection/embryo transfer cycles.A total of 72 women following ovarian stimulation and intracytoplasmic sperm injection were included. Blood serum samples were drawn at the day of chorionic gonadotropin administration. Follicular fluids were collected at the day of oocyte retrieval. The total fractions of tumor necrosis factor alpha, interleukin (IL)-1beta and IL-6 were measured with commercially available immunoassays.The concentrations of IL-1beta, both in serum and follicular fluids, were significantly different between ICSI cycles that resulted in pregnancy and those that failed. The concentrations of the other two cytokines did not significantly differ between successful and unsuccessful cycles.The circulating and intrafollicular concentrations of IL-1beta seem to be related to the pregnancy outcome in ICSI cycles of healthy women.

This study investigated the serum levels of resistin, adiponectin and leptin during the physiological menstrual cycle. Sixteen women (age: 19-30 years; body mass index: 19.46-24.9) with regular menstrual cycles participated. Fasting blood samples were collected on alternate days throughout a full menstrual cycle. Mean resistin concentrations were slightly higher during the luteal phase (5.30+/-0.23 ng/ml) compared to the follicular (4.68+/-0.07 ng/ml) and midcycle (4.86+/-0.09 ng/ml) phases (p=0.032). Mean leptin concentrations during the follicular phase (18.14+/-0.28 ng/ml) were significantly lower compared to the midcycle (21.79+/-0.29 ng/ml, p=0.006) and luteal phases (23.75+/-0.64 ng/ml, p

The recently identified gastric hormone ghrelin was initially described as a natural Growth Hormone Secretagogue Receptor ligand. Apart from ghrelin's first discovered action, which was the stimulation of Growth Hormone release, implications for many other functions have been reported. It seems that ghrelin exhibits an important role in conditions related to processes regulating nutrition, body composition and growth, as well as heart, liver, thyroid or kidney dysfunction. In this review, current available knowledge about ghrelin's role in various pathological conditions is presented.

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.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.Of the processed semen samples 17.85% hador =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.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.

Gonadotropin-releasing hormone (GnRH) plays a pivotal role in the physiology of reproduction in mammals. GnRH acts by binding to the GnRH receptor (GnRHR). In humans, only 1 conventional GnRH receptor subtype (type I GnRH receptor) has been found. In the human genome, 2 forms of GnRH have been identified, GnRH-I (mammal GnRH) and GnRH-II (chicken GnRH II). Both forms and their common receptor are expressed, apart from the hypothalamus, in various compartments of the human ovary. Gonadal steroids, gonadotropins, and GnRH itself controls the regulation of the GnRH/GnRHR system gene expression in the human ovary. The 2 types of GnRH acting paracrinally/autocrinally influence ovarian steroidogenesis, decrease the proliferation, and induce apoptosis of ovarian cells. In this review, the biology of GnRH/GnRHR system in humans, the potential roles of GnRH, and the direct effects of GnRH analogues in ovarian cells are discussed.

SUMMARY 1 Erythropoietin (EPO) is a hormone regulating the proliferation and differentiation of erythroid precursor cells. The hypothesis that haematopoietic and endothelial cells share a common haemanglioblast progenitor among others is based on the finding that both cell lineages express cell surface antigens, such as CD31 and CD34. 2 In the present study, we investigated the angiogenic potential of recombinant human erythropoietin on cyclosporine A (CsA)-induced nephrotoxicity in the rat kidney and compared it with the effect of basic fibroblast growth factor (bFGF), a well-known angiogenic factor. 3 Rats were divided into five groups: A (control), B (EPO treated), C (CsA treated), D (CsA + EPO treated) and E (CsA + bFGF treated). Mouse anti-human CD31 and CD34 antibodies were used to evaluate the kidney vessels present in histological preparations. 4 Glomerular and peritubular capillaries in Group B (EPO) were increased compared with the control (Group A; P

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 aim of this study was to investigate the concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), leptin, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-6, in cycles with a premature rise of serum progesterone. 25 intracytoplasmic sperm injection (ICSI) cycles with (Group 1) and 25 ICSI cycles without a premature progesterone elevation (Group 2) were included. The cut-off value of serum progesterone on the day of human chorionic gonadotropin (hCG) administration was 0.9 ng/ml. The indication for ICSI was male factor infertility exclusively. On the day of hCG injection, serum IL-6, VEGF and bFGF were significantly higher in Group 1 (7.7+/-24.5 pg/ml, 290.2+/-161.4 pg/ml and 15.7+/-8.2 ng/ml respectively) than in Group 2 (1.7+/-0.7 pg/ml, 175.2+/-92.1 pg/ml, and 9+/-1.6 ng/ml respectively). On the day of follicular puncture, serum cytokine concentrations were similar in the two groups. IL-6 intrafollicular concentrations were higher in Group 1 (14.7+/-20.7 pg/ml) than in Group 2 (9+/-9.3 pg/ml, p=0.031). There were no differences regarding the ICSI outcome. Patients with serum progesterone above 0.9 ng/ml, have elevated serum concentrations of IL-6, VEGF, and bFGF, as well as elevated intrafollicular concentrations of IL-6. The outcome of ICSI cycles is not associated with premature elevation of progesterone when the cut-off value is set at 0.9 ng/ml.

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.

Objective To evaluate the secretion of E 2 , P, and vascular endothelial growth factor (VEGF) in human granulosa luteinized cell cultures with the presence of a gonadotropin-releasing hormone (GnRH) agonist or antagonist. Design In vitro cell culture study. Setting Research laboratory of a university hospital. Patients Granulosa luteinized cells were obtained from 24 patients undergoing ovarian stimulation for IVF treatment. Interventions Granulosa cells were cultured for 48 hours with 1 nM of cetrorelix or leuprorelide. For a further 48 hours, granulosa cells were cultured with or without the combination of cetrorelix plus leuprorelide. Main Outcomes At the end of each culturing period, the concentrations of E 2 , P, and VEGF were measured in culture supernatants by immunoassays. Results Estradiol and P concentrations were similar between the culture supernatants from controls and treatment groups. The VEGF concentrations in supernatants from cultures with cetrorelix (2,315.1 ± 1,565.5 pg/mL) were moderately, but significantly, lower than in controls (2,604.3 ± 1,907.1 pg/mL) or cultures with leuprorelide (2,558.8 ± 1,403.1 pg/mL). Conclusions The GnRH analogues do not affect steroidogenesis in human granulosa luteinized cell cultures. The GnRH antagonists moderately affect the secretion of VEGF from human granulosa luteinized cells.

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.

For a successful fertilization and pronucleus formation to occur, not only a properly condensed sperm nucleus, but also decondensation ability in the oocyte is important. The sperm abnormalities causing failure of sperm decondensation in the oocyte are unrecognizable by conventional semen analysis and different methods are used. The chromatin decondensation ability of the human spermatozoa in vivo and in vitro and its association with infertility and assisted reproduction techniques (ART) are clearly discussed in this paper. The factors affecting the decondensation ability of the human sperm are also mentioned. It is suggested that the methods currently used to assess sperm chromatin decondensation are of limited value in assessing fertilization and pregnancy rates after ART.

Although angiogenetic therapy using recombinant growth factors holds much hope for the treatment of ischemic diseases, there are still unanswered questions including the method, doses or duration of therapeutic approach. We evaluated the angiogenetic effects of vascular endothelial growth factor (VEGF) on rat heart and gastrocnemius muscles when this was administered intramuscularly and compared them to those obtained from rats, which exercised daily.Both daily swimming exercise and intramuscular administration of VEGF increased angiogenesis in rat heart, even though exercise alone was the only one that increased angiogenesis quite significantly. The combined protocol (administration of growth factor and exercise) led to an increase of angiogenesis in cardiac muscles. In contrast, there was no effect on the lateral gastrocnemius muscle either by VEGF or exercise, whereas these together induced angiogenesis locally at the site of injection.

Cytokines play an important but controversial role during ovarian folliculogenesis for the development of mature and fertilizable oocytes. In this study, leptin, interleukin-1beta (IL1beta), tumor necrosis factor-alpha (TNFalpha) and vascular endothelial growth factor (VEGF) in serum and follicular fluids (FF) of women undergoing ovarian hyperstimulation were evaluated as prognostic markers of the outcome of intracytoplasmic sperm injection (ICSI) cycles.Ninety-five ICSI cycles were included in the study. The cytokines were measured in serum and FF samples with enzyme immunoassay methods.The cytokine concentrations in serum were not significantly correlated with the cytokine concentrations in FFs. Serum IL1beta was inversely-correlated with the number of retrieved oocytes. Serum TNFalpha was negatively-correlated with fertilization rate. In FFs, TNFalpha was positively-correlated with leptin. Leptin and VEGF in FFs were negatively-associated with pregnancy outcome.Leptin and VEGF concentrations in FFs may serve as prognostic markers of success after ovarian hyperstimulation and ICSI.

Ovarian hyperstimulation syndrome (OHSS) is a frequent serious complication in controlled ovarian hyperstimulation for in vitro fertilization. Among the treatments for OHSS, the continuation of GnRH agonist during the luteal phase has been recently suggested.Five patients at risk for OHSS received 0.1 mg triptorelin for one week after embryo transfer in order to prevent OHSS.All the five patients developed mild symptoms. None of them required hospitalization.Although the pathophysiology of OHSS and the effects of GnRH agonist during the luteal phase remain unclear, the continuation of GnRH agonist seems to be a safe and effective treatment for OHSS.

Triidothyronine(T3) plays a critical role in the physiological function of virtually all tissues. In the present study the effect of T3 on nerve regeneration was studied.The right sciatic nerve of 20 male rabbits was axonotomised. Ten of the animals were used as controls while the others received T3. Seven days later, all were sacrificed. Both sciatic nerves were excised and each axonotomised nerve was divided into three equal segments: I (proximal to the lesion), II (containing the lesion), III (distal to the lesion). Corresponding segments from the contralateral nerves were used as controls. After subcellular fractionation, proteins were analyzed by polyacrylamide gel electrophoresis and identified by proteomic analysis.T3 administration increased the levels of tubulins alpha 6 and beta 3, beta-actin, myelin protein zero, myelin basic protein, myelin protein-2 and histones H3 and H2A in segments I and III of the injured nerves. Increases in the levels of beta-actin and Y box-binding protein correlated to the event of injury rather than T3 administration.Our findings indicate that T3 treatment positively affects peripheral nerve regeneration.

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.