Evaluating the value of day 0 of an ICSI cycle on indicating laboratory outcome.

A number of oocyte characteristics have been associated with fertilization, implantation and live-birth rates, albeit without reaching a consensus. This study aims to delineate possible associations between oocyte characteristics, oocyte behavior during intracytoplasmic sperm injection (ICSI), fertilization potential, and laboratory outcomes. Four-hundred and seventy-seven patients, yielding 3452 oocytes, were enrolled in this prospective observational study from 2015 to 2018. Οoplasm granularity was associated with poor embryo quality and higher probabilities of post-ICSI oocytes and embryos discarded in any developmental stage and never selected for embryo transfer or cryopreservation (p < 0.001). Both sudden or difficult ooplasm aspiration, and high or lack of resistance during ICSI were associated with either a poor Zygote-Score or fertilization failure (p < 0.001). Sudden or difficult ooplasm aspiration and high resistance during ICSI penetration were positively associated with resulting to a post-ICSI oocyte or embryo that would be selected for discard. Evaluation of oocyte characteristics and oocyte behavior during ICSI may provide early information regarding laboratory and cycle outcomes. Particularly, ooplasm granularity, and fragmentation of polar body, along with sudden or difficult ooplasm aspiration and high or lack of resistance during ICSI penetration may hinder the outcome of an ICSI cycle. The associations presented herein may contribute towards development of a grading system or a prediction model. Taking into account information on oocytes and ICSI behavior may effectively assist in enhancing IVF outcome rates.

The use of serum anti-Mullerian hormone (AMH) levels and antral follicle count (AFC) to predict the number of oocytes collected and availability of embryos for cryopreservation in IVF.

AIM: To investigate the predictive value of anti-Mullerian hormone (AMH) and antral follicle count (AFC) on the final number of oocytes retrieved and the availability of embryos for cryopreservation in in vitro fertilization (IVF) cycles. PATIENTS AND METHODS: In this prospective study, one hundred and twenty women in their first IVF treatment were enrolled. The short stimulation agonist protocol was used for controlled ovarian hyperstimulation in all cases. Serum AMH levels were measured during the menstrual cycle preceding treatment. AFC was measured in cycle day 2, just before starting ovarian stimulation. RESULTS: A strong, positive correlation between AMH, AFC and the number of collected oocytes was found. The patients with available and suitable supplementary embryos for cryopreservation had higher levels of AMH and larger numbers of AFC. CONCLUSION: AMH and AFC appear to be valuable markers mainly for ovarian reserve and response to IVF treatment. Serum AMH levels and AFC are significantly associated with the number of retrieved oocytes. Also, a positive correlation with the availability of supernumerary embryos suitable for cryopreservation was observed.

Follicular fluid levels of vascular endothelial growth factor and its receptors and pregnancy outcome of women participating in intracytoplasmic sperm injection cycles.

PURPOSE: The intracytoplasmic sperm injection (ICSI) outcome is depended mainly on oocyte quality. Cytokines and their receptors play a critical role in oocyte maturation, fertilization, and embryo implantation. The purpose of the study was to study the levels of vascular endothelial growth factors (VEGFA, VEGFR1, VEGFA) in follicular fluids (FF) women participating in ICSI-in vitro fertilization (IVF) cycles in relation to cycle's outcome. MATERIAL AND METHODS: One hundred and fifty three samples of 70 women participating in ICSI cycles were classified in three infertility groups: male factor, female factor, and low responders. For controlled ovarian stimulation in male and female factor group, the long agonist protocol with leuprolide and recombinant follicle stimulating hormone (FSH) was employed, while the antagonist cetrorelix was used in low responders. Cytokines levels were evaluated with enzyme-linked immunosorbent assay (ELISA). RESULTS: In a total of 153 samples, the overall pregnancy rate was 51.6%, the higher one observed in female factor group (59% vs. 37.5% and 28.6% in male a factor and low responders group, p = 0.013. VEGFR2 differed statistically significantly between the two groups, being higher in the pregnancy group [median (IQR): 5,630 (4,870 - 6,651) vs. 4938 (4,068 - 6,020) in the non-pregnancy group, p = 0.003]. There were significant correlations between VEGF receptors, differentiated depending on infertility groups. CONCLUSIONS: The VEGFA/VEGFR2 system is important in human reproduction and the association pattern between VEGFA receptors may serve as a marker for ICSI outcome. Examination for spermatozoa functional defects may increase pregnancy rate in male factor group.

Sperm nuclear DNA fragmentation and its association with semen quality in Greek men.

Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n = 184) and non-normozoospermic (n = 485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non-normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility.

Predictive value of anti-müllerian hormone, follicle-stimulating hormone and antral follicle count on the outcome of ovarian stimulation in women following GnRH-antagonist protocol for IVF/ET.

AIM: To compare the efficacy of three methods: the levels of anti-müllerian hormone (AMH), the levels of follicle stimulating hormone (FSH) and the antral follicle count (AFC), for the prediction of the number of retrieved mature oocytes and the number of generated embryos by intracytoplasmic sperm injection (ICSI) in women stimulated with a GnRH-antagonist protocol. MATERIALS AND METHODS: 105 women were enrolled in the study. At the day 2 of a preceding cycle, AFC was performed and FSH and AMH were measured in serum by immunoenzymatic assays. All women were stimulated with a GnRH-antagonist protocol and ovulation was induced with human chorionic gonadotropin. ICSI was performed in all retrieved mature oocytes. Embryo transfers were performed at days 2-3. According to the oocytes retrieved, patients were categorized as poor (<4), normal (4-12) or high responders (>12). RESULTS: AFC and the levels of baseline FSH and AMH were significantly different among poor, normal and high responders. The number of oocytes as well as the number of embryos was negatively correlated with baseline FSH and positively correlated with baseline AMH and AFC, whereas AFC showed the strongest correlation. Stepwise regression analysis indicated AFC and baseline AMH as the most significant parameters for the prediction of the number of oocytes; for the prediction of the number of embryos, the most significant parameter was AFC. CONCLUSIONS: AFC, baseline AMH and baseline FSH are good predictors for the outcome of ovarian stimulation in GnRH-antagonist cycles. However, AFC appears to have the best predictive value.

Serum pattern of circulating soluble receptor of leptin throughout the menstrual cycle.

AIM: The aim of the study was to investigate the pattern of circulating soluble receptor of leptin (sLeptinR) during the menstrual cycle and the association of sLeptinR to leptin, sex hormones and gonadotropins. METHODS: Fasting blood samples were collected on alternate days throughout a full cycle from fifteen healthy volunteers. Immunoenzymatic assays were employed to record the relevant levels. RESULTS: sLeptinR concentrations throughout the cycle were found to vary negligibly. No significant correlations between sLeptinR and leptin, gonadotropins or progesterone, were established. During the follicular phase, subjects presenting with higher estradiol levels tended to have higher sLeptinR concentrations. CONCLUSION: The little variation of sLeptinR concentrations during the menstrual cycle indicates that the rise of leptin during the luteal phase implies an increase of its bioactivity.

Distribution of ABO and Rh blood groups in Greece: an update.

The aim of this study was to investigate and evaluate the frequency of the antigens classifying the ABO and Rh blood groups in the Greek population. In this study the 3.5% were first generation immigrants with both their parents immigrants from countries of the USSR, while 1.2% had only one immigrant parent, while the other one was Greek. We compared the frequency of distribution of blood groups ABO and Rh to previous studies conducted at a time before Greece became destination for refugees and immigrants from East and Northeast countries. Blood samples were collected from first year medical students. The frequency of distribution of the ABO and Rh blood groups was slightly differentiated in comparison to previous relevant studies. Significant increase was recorded with respect to the emergence of blood group B in the population investigated, and a considerable reduction was noted in blood group O. In reference to the remaining blood groups, no statistically significant difference was documented. The genetic pool and the genetic inventory of the population residing in Greece have been modified during the last years potentially due to the first generation immigrants. The results of this study could contribute significantly to the National Health System in aiding the prediction of percussions of certain diseases related to blood groups, as well as the requirement for certain blood groups within the blood donation program.

Cytokine profile in cases with premature elevation of progesterone serum concentrations during ovarian stimulation.

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.

Modulatory role of adenosine and its receptors in epilepsy: possible therapeutic approaches.

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.

Comet assay of cumulus cells and spermatozoa DNA status, and the relationship to oocyte fertilization and embryo quality following ICSI.

It has been postulated that apoptosis may affect cumulus cell and sperm DNA integrity, and therefore influence the outcome of assisted reproductive techniques. This study investigates apoptotic levels in both cumulus cells and spermatozoa, and their relationship with fertilization and embryo quality after intracytoplasmic sperm injection (ICSI). The neutral comet assay was performed on cumulus cells and semen samples from 55 couples with male factor infertility undergoing ICSI treatment. Cells were fixed in agarose on comet assay slides, lysed in a neutral buffer and submitted to electrophoresis. The cells were stained with SYBR green fluorescent dye, which binds to double-stranded DNA and upon excitation emits light. Analysis showed that there was no correlation between apoptosis levels and the outcome of ICSI (fertilization and embryo quality).

IGF-I and epidermal growth factor levels in follicular fluid of women undergoing controlled ovarian hyperstimulation using the multidose GnRH-antagonist protocol or the long GnRH-agonist protocol.

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.

Angiogenic effect of intramuscular administration of basic and acidic fibroblast growth factor on skeletal muscles and influence of exercise on muscle angiogenesis.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: Significant increases in the numbers of blood vessels of the right gastrocnemius muscles in groups C1 and D1 were observed compared with controls (p<0.05). There was only a slight increase in angiogenesis in the left gastrocnemius muscle of groups C1 and D1 compared with controls (p>0.05), and there was a decrease in angiogenesis in the gastrocnemius muscle of the swimming group compared with controls. CONCLUSION: The intramuscular administration of b-FGF, but not a-FGF, induced significant local angiogenesis in gastrocnemius muscle at the site of injection.

Follicular fluid levels of vascular endothelial growth factor and leptin are associated with pregnancy outcome of normal women participating in intracytoplasmic sperm injection cycles.

Cytokines play a critical and multifarious role in follicular maturation. Consequently, they may influence the pregnancy outcome in cycles of assisted reproduction. The aim of this study was to measure the levels of tumor necrosis factor-alpha (TNFalpha), vascular endothelial growth factor (VEGF) and leptin in serum and follicular fluids (FFs) from women undergoing controlled ovarian hyperstimulation (COH) for intracytoplasmic sperm injection cycles (ICSI). We tried to investigate their interrelationships and to evaluate them as predictive markers for the cycle's outcome. Seventeen women participated in this study. Male factor infertility was the only indication for ICSI cycles. For COH, the long agonist protocol with triptorelin and recombinant FSH was employed. Cytokines levels were evaluated by ELISA. Serum cytokine levels did not differ between pregnant and non-pregnant women. FF-VEGF levels were significantly elevated in non-pregnant women (722.2+/-1093.2 pg/ml) as compared to pregnant women (290.3+/-259.8 pg/ml). Leptin concentrations were also significantly higher in FFs of non-pregnant women (682.6+/-625.1 ng/ml) than those of pregnant women (231.6+/-286.5 ng/ml). There were significant positive correlations between FF-leptin and age, as well as between FF-leptin and FF-VEGF concentrations. It was concluded that elevated FF-leptin and VEGF levels are associated with failure of conception in IVF cycles and may serve as markers in clinical practice.

A borderline form of empty follicle syndrome. Case report.

BACKGROUND: Empty follicle syndrome is known as the failure of oocyte retrieval despite the adequate response to ovarian stimulation. It is a rare phenomenon in in-vitro fertilization and borderline forms of this syndrome have also been described. MATERIALS AND METHODS: Two cycles in the same patient were stimulated with GnRH agonist/hMG and recFSH; the first followed the long and the second followed the short protocol. RESULTS: There was a sudden drop in estradiol levels while the ovaries contained a large number of small and medium sized follicles. hCG was administered and oocyte retrieval was performed 36 hours later. There was no indication of low hCG levels. For the first cycle two oocytes were collected: one degenerated and one of poor quality. The second cycle resulted in total failure of oocyte retrieval. CONCLUSION: The two cycles were classified as borderline forms of empty follicle syndrome. The possible aetiology is discussed.

Triptorelin versus cetrorelix in intracytoplasmic sperm injection cycles in women with a single ovary.

OBJECTIVE: To compare the response to ovarian stimulation with either the long protocol of a GnRH-agonist or the multiple protocol of a GnRH-antagonist, in women with a single ovary who underwent intracytoplasmic sperm injection (ICSI) cycles. STUDY DESIGN: Retrospective study including 75 ICSI cycles from 26 women. Sixty-three cycles were stimulated with triptorelin/hMG or rFSH, whereas 12 cycles were stimulated with cetrorelix/hMG or rFSH. RESULTS: There was not found any statistical significant difference between the two groups regarding the days of stimulation, the number of gonadotropins' ampoules, the peak estradiol levels, the number of aspirated follicles and the number of retrieved oocytes. The fertilization rate, the number of transferred embryos as well as the cumulative embryo score were also similar in both groups. CONCLUSION: The multiple stimulation protocol of cetrorelix is equally effective with the long protocol of triptorelin in the ovarian stimulation of women with a single ovary.

Gonadotropin releasing hormone antagonists and cryopreservation outcome: a review.

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. CONCLUSION: 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.

Father-to-son sperm donation. A report of three cases.

Sperm donation is a common practice in assisted reproduction. In cases of azoospermia and negative results of testicular sperm extraction, it appears as the only solution. Sperm donation entails a complete genetic dissociation between husband and offspring, which brings psychological stress for the couple arising from ethical and existential dilemmas. Faced with such dilemmas, some couples prefer father-to-son donation as an alternative solution. Here, three cases of non-obstructive azoospermia are presented where intracytoplasmic sperm injection was performed with father-to-son sperm donation.

A comparison of the ovarian response to external stimulation between women with right or left ovary participating in programs of ICSI/ET.

INTRODUCTION: Single ovary women compose a group of special interest in assisted reproduction technologies. The aim of the present study was to investigate whether the response to external stimulation and the outcome in women with a single ovary was affected by the location of the ovary. METHODS: Sixty cycles from 24 women, which stimulated with either hMG or rFSH and a GnRH agonist, were retrospectively studied. Twelve women had right and 12 had left ovary. In all cases, the fertilization was performed by intracytoplasmic sperm injection. RESULTS: There was not found any statistical significant difference between the two groups regarding the days of stimulation, the peak estradiol levels, the number of aspirated follicles, the number of retrieved oocytes, the fertilization rate and the number of transferred embryos. The cumulative embryo score (CES) was also similar in both groups. Six pregnancies in the group of the right ovary and seven in the group of left ovary were achieved. CONCLUSION: According to these results, right and left ovary, in single ovary women under external stimulation, are equally active and potent for reproduction.

Outcome of anticipated ICSI cycles using intentionally frozen-thawed testicular spermatozoa according to the spouse's response to ovarian stimulation.

PURPOSE: To investigate the outcome of ICSI cycles, using frozen-thawed testicular spermatozoa from patients suffering from non-obstructive azoospermia, in relation to the spouse's response to ovarian stimulation. METHODS: A retrospective study with two groups of couples where males suffered from non-obstructive azoospermia. In group 1 (n = 14), the female partners responded poorly to ovarian stimulation (< or = 4 oocytes retrieved). In group 2 (n=14), the female partners responded well (> or = 10 oocytes retrieved). Both groups underwent 14 cycles of ICSI using frozen-thawed spermatozoa obtained by TESE. RESULTS: The total amount of gonadotropin, the duration of stimulation, the peak estradiol concentrations and the number of oocytes were significantly different between the two groups. Despite the satisfactory fertilisation rates, the outcome in poor responders was disappointing due to a low number of oocytes. There was only one pregnancy in the poor responder group whereas there were four in the group that responded well. The pregnancy rates per oocyte collection were 7.14% in group 1 versus 28.57% in group 2. The implantation rates were 60.60% versus 55.33%, respectively. CONCLUSION: It is possible to achieve satisfactory fertilisation rates using frozen-thawed, surgically-retrieved testicular spermatozoa, but the poor ovarian response to stimulation induction is the limiting factor in reaching implantation and pregnancy. It is preferable that poorly stimulated cycles be canceled, in the hope of a better subsequent response.

Comparison of poor responders with good responders using intentionally frozen-thawed epididymal spermatozoa in subsequent ICSI cycles.

PURPOSE: To compare the outcome of intracytoplasmic sperm injection (ICSI) cycles performed with frozen-thawed epididymal spermatozoa between patients who respond poorly and patients who respond well to ovarian stimulation. METHODS: 17 patients suffering from obstructive azoospermia underwent microsurgical retrieval of epididymal spermatozoa (MESA) and the spermatozoa were frozen. The frozen-thawed spermatozoa were used in subsequent ICSI cycles. In six patients, the female partners responded poorly to ovarian stimulation. They accomplished nine ICSI cycles. In 11 patients, the female partners responded well to ovarian stimulation and they accomplished 16 cycles. RESULTS: Poor responders were older than those who reponded well. The mean number of metaphase II oocytes collected was lower in the poor responder group. In the poor responders, two couples failed to fertilise the oocytes in two ICSI cycles. In the good responders, one couple failed to fertilise the oocytes in an ICSI cycle. There were no significant differences in fertilization rates between the two groups. The estradiol concentrations on the day of hCG administration were significantly higher in the good responders. There was no pregnancy in the poor responder group, while three patients who responded well conceived. Eight good responders had 34 supernumerary 2PN oocytes which were cryopreserved. CONCLUSION: Frozen-thawed epididymal spermatozoa from men with obstructive azoospermia are potent to achieve satisfactory fertilization rates. Poor ovarian response to stimulation induction appears to be the main limiting factor in reaching the stage of embryo transfer. It is preferable in older women to cancel cycles with poor response in the hope that a better response might be obtained in a subsequent cycle. Thus, the frozen-thawed epididymal sperm can be preserved and the most stressful and expensive phase of IVF-ICSI treatment can be avoided.