Capsaicin protects against testicular torsion injury through mTOR-dependent mechanism.

SCOPE: Testicular torsion and subsequent release of reactive oxygen species (ROS) can cause infertility in adults. Oxidative stress following testicular torsion plays an important role in the ýonset and development of apoptotic cell death through dysregulation of the cellular signaling pathways. Anti-inflammatory and antioxidant properties of capsaicin, a bioactive composition present in red peppers, has already been exploited for treatment of the cancer and pain relief. In present work, we evaluated the role of the mammalian target of rapamycin (mTOR) in antioxidant effect of capsaicin against reperfusion injury following testicular torsion. METHODS: Male Wistar rats weighing 200-220 g were randomly assigned into four major groups: (i) a sham operated group, (ii) a testicular torsion (TT) group, (iii) three groups treated with different doses of capsaicin (TT + 100, 500 and 1000 µg/ml Cap), and (iv) three groups of healthy rats treated with different doses of capsaicin (100, 500 and 1000 µg/ml). Western blotting assay was used to examine the anti-apoptotic effects of capsaicin in testicular cells following torsion. H&E and TUNEL methods were used to evaluate testicular morphology and apoptosis activity. RESULTS: Compared to control group, phosphorylation of mTOR was significantly increased in the TT groups. Capsaicin administration remarkably decreased the phosphorylation of mTOR at the highest dose (P < 0.05). Capsaicin decreased apoptosis and preserved tubular morphology in testes. CONCLUSION: Our results showed that antioxidant properties of capsaicin minimizes cell death and reperfusion injury following testicular torsion.

Ooplasmic transfer in human oocytes: efficacy and concerns in assisted reproduction.

BACKGROUND: Ooplasmic transfer (OT) technique or cytoplasmic transfer is an emerging technique with relative success, having a significant status in assisted reproduction. This technique had effectively paved the way to about 30 healthy births worldwide. Though OT has long been invented, proper evaluation of the efficacy and risks associated with this critical technique has not been explored properly until today. This review thereby put emphasis upon the applications, efficacy and adverse effects of OT techniques in human. MAIN BODY: Available reports published between January 1982 and August 2017 has been reviewed and the impact of OT on assisted reproduction was evaluated. The results consisted of an update on the efficacy and concerns of OT, the debate on mitochondrial heteroplasmy, apoptosis, and risk of genetic and epigenetic alteration. SHORT CONCLUSION: The application of OT technique in humans demands more clarity and further development of this technique may successfully prove its utility as an effective treatment for oocyte incompetence.

Reconstruction of mammalian oocytes by germinal vesicle transfer: A systematic review.

Nuclear transfer procedures have been recently applied for clinical and research targets as a novel assisted reproductive technique and were used for increasing the oocyte activity during its growth and maturation. In this review, we summarized the nuclear transfer technique for germinal vesicle stage oocytes to reconstruct the maturation of them. Our study covered publications between 1966 and August 2017. In result utilized germinal vesicle transfer techniques, fusion, and fertilization survival rate on five different mammalian species are discussed, regarding their potential clinical application. It seems that with a study on this method, there is real hope for effective treatments of old oocytes or oocytes containing mitochondrial problems in the near future.

Experimental strategies towards increasing intracellular mitochondrial activity in oocytes: A systematic review.

PURPOSE: The mitochondrial complement is critical in sustaining the earliest stages of life. To improve the Assisted Reproductive Technology (ART), current methods of interest were evaluated for increasing the activity and copy number of mitochondria in the oocyte cell. METHODS: This covered the researches from 1966 to September 2015. RESULTS: The results provided ten methods that can be studied individually or simultaneously. CONCLUSION: Though the use of these techniques generated great concern about heteroplasmy observation in humans, it seems that with study on these suggested methods there is real hope for effective treatments of old oocyte or oocytes containing mitochondrial problems in the near future.

Using Fresh and Frozen Testicular Sperm Samples in Couples Undergoing ICSI-MicroTESE Treatment.

BACKGROUND: We performed this study to evaluate use of fresh and frozen sperm samples in non-obstructive azoospermia microdissection testicular sperm extraction (micro-TESE-ICSI) treatment. METHODS: We performed a total of 82 consecutive in vitro fertilization (IVF) cycles at Fertijin IVF Center in Istanbul, Turkey from January 2010 to March 2012. In 43 participants we used fresh sperm and frozen sperm in the remaining 39 cases. We used fresh and frozen thawed micro surgical testicular sperm extraction (micro TESE) sperm for ICSI with metaphase II (MII) oocytes. RESULTS: Frozen microTESE sperm was used in 39 cycles, while 43 ICSI cycles were performed using fresh microTESE. Neither the age of male partners (38.33±5.93 and 38.13±8.28) nor that of the female participants (33.16±6.38 and 33.33±6.97) showed significant difference between fresh versus the microTESE and frozen treatment groups, respectively. FSH concentrations were (14.66±13.93 mIU/ml) in fresh TESE group and (17.91±16.29 mIU/ml) in frozen group with no correlations or differences between the two groups. The average number of mature oocytes injected with sperm was 9.23±3.77, versus 9.26±5.26 in cycles using fresh and frozen microTESE sperm, respectively. Fertilization rate was not significantly different in the fresh microTESE (44.79%) than frozen TESE sperm group (46.76%). The average number of transferred embryos was 1.60±0.49 in fresh sperm group and 1.59±0.50 in frozen sperm group. All embryo transfers were performed on day 3. CONCLUSION: Cryopreservation of testicular sperm tissues is more suitable and of great benefite if carried out before ovulation induction and not after, especially in cases with non-obstructive azoospermia.

Clinical value of DNA fragmentation evaluation tests under ART treatments.

Male reproductive health has been under scrutiny recently. Many studies in the literature have concluded that semen quality is declining and that the incidence of testicular cancers is increasing. The reason for this change has been attributed to damage in sperm chromatin. During in vivo reproduction, the natural selection process ensures that only a spermatozoon with normal genomic material can fertilize an oocyte. However, the assisted reproduction technique (ART) is our selection process, leading to the possibility that abnormal spermatozoa could be used to fertilize an oocyte. We could avoid this by quantifying the amount and type of genomic damage in sperm using well-accepted laboratory methods. The sperm deoxyribonucleic acid (DNA) integrity is important for success of natural or assisted fertilization as well as normal development of the embryo, fetus and child. Intra cytoplasmic sperm injection (ICSI) is bypassing natural sperm selection mechanisms, which increases the risk of transmitting damaged DNA. The significance of required investigations and multiple techniques is that they could evaluate DNA defects in human spermatozoa. The ability of these techniques to accurately estimate sperm DNA damage depends on many technical and biological aspects. The aim of this review is to evaluate the most commonly used methods.

Emerging gonadotropin-releasing hormone agonists.

INTRODUCTION: Gonadotropin-releasing hormone agonist analogs (GnRHa) are peptides that mimic the action of gonadotropin-releasing hormone (GnRH) and are used to suppress subsequent sex steroid production. Although the analogs are a rather defined group of drugs, there have been developments in the past decades and there is still ample room for improvement. New therapeutic strategies in the use of GnRHs are discussed. AREAS COVERED: Major points of discussion include: i) the use of concomitant treatment of early breast cancer in premenopausal estrogen-positive and -negative patients, ii) the use of GnRHa for fertility preservation in young female patients with malignant diseases and iii) the use of GnRH analogs in assisted reproduction. The manuscript provides a better understanding of GnRH agonists as well as an explanation of their major indications, biochemical pathways and concluding therapeutic strategies. Recent results from international meetings and debates are described to explain current controversies. EXPERT OPINION: This paper highlights the need for more complex GnRH analogs. In the next few years, there will be longer acting GnRHas that may improve adherence. New therapeutic targets in oncological concepts may go beyond fertility preservation and focus on the antiproliferative effects of GnRH analogs.

Concentrations of inflammatory cytokines and the outcome in ICSI cycles.

UNLABELLED: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: The circulating and intrafollicular concentrations of IL-1beta seem to be related to the pregnancy outcome in ICSI cycles of healthy women.

Intrafollicular and circulating concentrations of leptin do not predict the outcome in IVF-ICSI cycles.

Leptin is involved not only in the regulation of food intake but also in other functions including reproduction. Because leptin has been demonstrated to influence ovarian steroidogenesis directly and leptin levels vary during the menstrual cycle and in stimulated cycles, we tested the hypothesis that serum or intrafollicular concentrations of leptin would correlate with reproductive outcomes in intracytoplasmic sperm injection cycles. Serum and follicular fluid samples were collected from 77 women undergoing ovarian stimulation, intracytoplasmic sperm injection and embryo transfer due to male factor infertility. The concentrations of total leptin, both in serum and in pooled follicular fluid samples, did not correlate with the number of oocytes, the fertilization rate or the embryo quality. Additionally, leptin concentrations did not differ between cycles that resulted in pregnancy and those that failed. These results raise objections to the prognostic value of leptin for the outcome of in vitro fertilization/intracytoplasmic sperm injection cycles.

A live birth after transfer of a day 2 embryo derived from frozen-thawed zygotes that had undergone polar body biopsy: a case report.

OBJECTIVE: To present a live birth after freezing and thawing of biopsied oocytes. DESIGN: Case report. SETTING: Artificial reproduction unit of a university hospital. PATIENT(S): A primary infertile couple with asthenoteratozoospermia and repeated failures of intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Screening of aneuploidy during the fourth ICSI cycle with polar body biopsy (PB) for repeated failures of artificial reproductive techniques and a transfer of a cryopreserved day 2 embryo derived from cryopreserved zygotes with slow-rate freezing after PB. MAIN OUTCOMES MEASURE(S): Live birth, viability, and survival. RESULT(S): A successful pregnancy and a live birth were presented after a transfer of day 2 embryos derived from oocytes that underwent PB and subsequent cryopreservation. CONCLUSION(S): Pregnancy can be obtained subsequent to cryopreservation and thawing after PB.

The levels of steroid hormones and cytokines in individual follicles are not associated with the fertilization outcome after intracytoplasmic sperm injection.

OBJECTIVE: To investigate the association between the levels of two steroid hormones and eight cytokines in fluids from individual follicles and the fertilization outcome of the oocytes derived from the same follicles. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Forty-three women participating in intracytoplasmic sperm injection (ICSI)/ET cycles. INTERVENTION(S): The ovarian stimulation followed the multidose GnRH antagonist protocol. ICSI was performed in mature oocytes. The concentrations of estradiol, progesterone, tumor necrosis factor-alpha, interleukin (IL) -1beta, IL-6, vascular endothelial growth factor, leptin, basic fibroblast growth factor, epidermal growth factor, and insulin-like growth factor-I were measured by immunoassay methods in the follicles from which the mature oocytes were derived. MAIN OUTCOME MEASURE(S): The concentrations of the above hormones and cytokines in individual follicles and the fertilization outcome of the oocytes derived from the same follicles. RESULT(S): The intrafollicular concentrations of the above factors were not significantly associated with the fertilization outcome. These factors were not correlated with embryo quality, with the exception of leptin, which was weakly associated with embryo score (R = 0.276). CONCLUSION(S): The intrafollicular concentrations of the above factors cannot predict the fertilization outcome after ICSI.

Matrix metalloproteinase (MMP)-2 and MMP-9 in seminal plasma.

PURPOSE: 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. METHODS: Basic semen analysis was performed in 82 semen samples. Seminal plasma was analyzed with gelatin zymography. RESULTS: 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 concentration > or = 50 x 10(6)/ml. CONCLUSION: 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.

Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing?

Cryopreservation of human oocytes and embryos is a necessary tool in assisted reproduction treatment that leads to an increased cumulative outcome while decreasing costs. Vitrification is a cryopreservation technique that leads to a glass-like solidification, with rapid cooling of cells or tissues. Nowadays vitrification is claimed to be the future of cryopreservation of human embryos due to improved survival rates and clinical outcomes. This study was conducted at a university clinic to assess the safety and efficiency of vitrification of human zygotes as a routine procedure. A total of 849 pronuclear-stage (PN) zygotes were vitrified between March 2004 and July 2006. During this period, 103 cycles of cryopreserved embryo transfer were completed. In total, 339 PN zygotes were thawed resulting in an 89% survival rate (302 PN zygotes). The mean number of embryos per transfer was 2.2. The pregnancy rate obtained was three times higher (36.9%) than that obtained with the slow-rate freezing method (10.2%) used previously in the same centre. In conclusion, vitrification of human zygotes at the pronuclear stage seems to be a successful and reliable method with favourable outcomes and can be recommended as a routine technique for cryopreservation of human embryos.

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.

Distribution of persistent organochlorine contaminants in infertile patients from Tanzania and Germany.

PURPOSE: To test whether environmental pollutants could affect fertility in humans. METHODS: 31 women and 16 men from Tanzania and 21 couples from Germany were included (n = 89). Pesticides and polychlorinated biphenyls were measured in serum, follicular fluid or seminal plasma by gaschromatography and related to sperm quality and pregnancy rates. RESULTS: Higher concentrations of DDT+DDE and dieldrin in Tanzania and higher concentrations of PCBs in Germany and in men were detected. All compounds showed higher concentrations in serum and lowest concentrations in seminal plasma. A lower pregnancy rate in German women with high serum concentrations of DDT+DDE was observed. The toxins had no impact on sperm quality. CONCLUSIONS: The distribution of toxins between agricultural and industrial countries is different. Seminal plasma seems to be inert against chemicals. In patients with high serum concentrations of DDT and DDE pregnancy rates were impaired.

Gonadotropin-releasing hormone antagonists do not influence the secretion of steroid hormones but affect the secretion of vascular endothelial growth factor from human granulosa luteinized cell cultures.

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.

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.

ICSI outcome is not associated with the incidence of spermatozoa with abnormal chromatin condensation.

BACKGROUND: The condensation of sperm chromatin during spermiogenesis and epididymal transport is of essential importance for fertilization. The main purpose of this study was to examine whether abnormalities of sperm nuclear condensation can influence the outcome of intracytoplasmic sperm injection (ICSI) cycles. MATERIALS AND METHODS: Semen samples from 154 ICSI cycles were studied. Before semen preparation for ICSI, basic semen analysis was performed and a small portion from each sample was fixed. The condensation of sperm nuclear chromatin was evaluated with chromomycin A3 under a fluorescence microscope. RESULTS: The incidence of spermatozoa with abnormal chromatin condensation was positively correlated with sperm concentration (p = 0.020565), but was not correlated with other semen parameters such as morphology and motility. Abnormal chromatin condensation was also not correlated with fertilization rate, cumulative embryo score or pregnancy rate. CONCLUSION: The above results indicate that ICSI outcome is not influenced by the incidence of spermatozoa with abnormal chromatin condensation.

Recombinant LH in ovarian stimulation.

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.

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.