Cryopreservation of in vitro matured oocytes in addition to ovarian tissue freezing for fertility preservation in paediatric female cancer patients before and after cancer therapy.

STUDY QUESTION: Is a protocol that combines in vitro maturation of germinal vesicle-stage oocytes and their vitrification with freezing of cortical ovarian tissue feasible for use in fertility preservation for both chemotherapy-naive paediatric patients as well as patients after initiation of cancer therapy? SUMMARY ANSWER: Follicle-containing ovarian tissue as well as oocytes that can undergo maturation in vitro can be obtained from paediatric patients (including prepubertal girls) both before and after cancer therapy. WHAT IS KNOWN ALREADY: Anticancer therapy reduces the number of follicles/oocytes but this effect is less severe in young patients, particularly the paediatric age group. Autotransplantation of ovarian tissue has yielded to date 60 live births, including one from tissue that was cryostored in adolescence. However, it is assumed that autografting cryopreserved-thawed ovarian cortical tissue poses a risk of reseeding the malignancy. Immature oocytes can be collected from very young girls without hormonal stimulation and then matured in vitro and vitrified. We have previously shown that there is no difference in the number of ovarian cortical follicles between paediatric patients before and after chemotherapy. STUDY DESIGN, SIZE, DURATION: A prospective study was conducted in a cohort of 42 paediatric females with cancer (before and after therapy initiation) who underwent fertility preservation procedures in 2007-2014 at a single tertiary medical centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study group included girls and adolescent females with cancer: 22 before and 20 after chemotherapy. Following partial or complete oophorectomy, immature oocytes were either aspirated manually ex vivo from visible small antral follicles or filtered from spent media. Oocytes were incubated in oocyte maturation medium, and those that matured at 24 or 48 h were vitrified. Ovarian cortical tissue was cut and prepared for slow-gradual cryopreservation. Anti-Mullerian hormone (AMH) levels were measured in serum before and after oophorectomy. MAIN RESULTS AND ROLE OF CHANCE: Ovarian tissue was successfully collected from 78.7% of the 42 patients. Oocytes were obtained from 20 patients before chemotherapy and 13 after chemotherapy. The youngest patients from whom oocytes were retrieved were aged 2 years (two atretic follicles) and 3 years. Of the 395 oocytes collected, ∼30% were atretic (29.6% in the pre-chemotherapy group, 37% in the post-chemotherapy group). One hundred twenty-one oocytes (31%) were matured in vitro and vitrified: 67.8% from patients before chemotherapy, the rest after chemotherapy. Mature oocytes suitable for vitrification were obtained from 16/20 patients before chemotherapy and from 12/13 patients after chemotherapy (maturation rate, 32 and 26.4%, respectively). There were significant correlations of the number of vitrified oocytes with patient age (more matured oocytes with older age) (P = 0.001) and with pre-oophorectomy AMH levels (P = 0.038 pre-chemotherapy group, P = 0.029 post-chemotherapy group). Oocytes suitable for vitrification were obtained both by manual aspiration of antral follicles (45%) and from rinse solutions after dissection. There were significantly more matured oocytes in the pre-chemotherapy group from aspiration than in the post-chemotherapy group after both aspiration (P < 0.033) and retrieval from rinsing fluids (P < 0.044). The number of pre-antral follicles per histological section did not differ in the pre- versus post-chemotherapy. AMH levels dropped by approximately 50% after ovarian removal in both groups, with a significant correlation between pre- and post-oophorectomy levels (P = 0.002 pre-chemotherapy group, P = 0.001 post-chemotherapy group). LIMITATIONS, REASONS FOR CAUTION: There were no patients between 5 years and 10 years old in the post-chemotherapy group, which might have affected some results and correlations. Oocytes from patients soon after chemotherapy might be damaged, and caution is advised when using them for fertility-restoration purposes. The viability, development capability and fertilization potential of oocytes from paediatric patients, especially prepubertal and after chemotherapy, are unknown, in particular oocytes recovered from the media after the tissue dissection step. WIDER IMPLICATIONS OF THE FINDINGS: Although more oocytes were collected and matured from chemotherapy-naïve paediatric patients, ovarian tissue and immature oocytes were also retrieved from young girls in whom cancer therapy has already been initiated. Our centre has established a protocol for potential maximal fertility preservation in paediatric female patients with cancer. Vitrified-in vitro-matured oocytes may serve as an important gamete source in paediatric female patients with cancer because the risk of reseeding the disease is avoided. Further studies are needed on the fertility-restoring potential of oocytes from paediatric and prepubertal patients, especially after exposure to chemotherapy. STUDY FUNDING/COMPETING INTERESTS: The study was conducted as part of the routine procedures for fertility preservation at our IVF unit. No funding outside of the IVF laboratory was received. Funding for the AMH measurements was obtained by a research grant from the Israel Science Foundation (to B.-A.I., ISF 13-1873). None of the authors have competing interests. TRIAL REGISTRATION NUMBER: N/A.

Perfusion with lipopolysaccharide differently affects the secretion of interleukin-1 beta and interleukin-1 receptor antagonist by term and preterm human placentae.

The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on the secretion of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) and of its natural inhibitor interleukin-1 receptor antagonist (IL-1Ra), by perfused human term and preterm placental tissue. Eight term and eight preterm placentae were collected immediately after delivery; four term and four preterm placentae were perfused with control medium (without LPS) and the other four term and four preterm placentae were perfused with medium containing LPS. The release of IL-1beta into the maternal compartment by term placenta was significantly higher than the release by preterm placenta (p<0.001). However, there were no significant differences between IL-1beta levels released into the fetal compartments of term and preterm placentae. No significant differences were observed in the release of IL-1Ra into the maternal and fetal compartments of term placenta, when compared to preterm placenta. Exposure to LPS significantly decreased the capacity of term placenta to release IL-1beta into the maternal compartment (p<0.001) and increased the capacity of term placenta to release IL-1Ra into the maternal and fetal compartments (p<0.001 and p=0.017, respectively). However, the capacity of preterm placentae to release IL-1beta and IL-Ra into the maternal and fetal compartments was not affected by LPS. IL-1beta was expressed by both term and preterm placentae before and after perfusion (+/- LPS), by epithelial cells of the amnion, chorion, by syncytiotrophoblast and stromal cells of villous tissue and by the decidua. IL-1Ra in term and preterm placentae was expressed before perfusion mainly in epithelial cells of the amnion. After perfusion of term placentae (+/- LPS), additional IL-1Ra expression was seen in epithelial cells of the amnion and in syncytiotrophoblast and stromal cells of villous tissue and by the decidua. However, perfusion of preterm placentae (+/- LPS) did not affect IL-1Ra expression. The localization of IL-1beta and IL-1Ra in both term and preterm human placental tissue suggests a their physiologic role. The data presented indicates that the IL-1 system in term and preterm placentae seems to be differently affected by LPS. Down-regulation in the release of the pro-inflammatory cytokine IL-1beta and the up-regulation of its antagonist (IL-1Ra) may be a part of the inflammatory response to infection in human term, but not preterm, placentae. The IL-1 system in term and preterm placentae seems to be differently affected by LPS.

Albumin-dependent digoxin transfer in isolated perfused human placenta.

OBJECTIVE: To determine the effects of albumin (BSA) concentration in perfusion medium on digoxin transfer in isolated perfused human placental cotyledon. STUDY DESIGN: Isolated placental cotyledons from 13 normal human placentas were dually perfused after cannulating artery and vein of the chorionic plate and piercing 4 catheters through the corresponding basal plate with M199 medium enriched with BSA and glucose. Flow rates were 12 and 6 ml/min in the maternal and fetal circuits, respectively. Digoxin was added to the maternal reservoir at a final concentration of 5.51 +/- 1.00 ng/ml. BSA in maternal and fetal perfusate was kept at 3 concentrations: 1, 3 and 5 mg/ml (Groups I, II, III). Transplacental passage of digoxin was calculated from repeated fetal and maternal perfusate samples collected over 3 hours in the 3 groups. Digoxin levels were measured by FPIA (TDx, Abbott). RESULTS: There was no transfer of digoxin from the maternal to fetal compartment when the concentration of BSA was 1 mg/ml. Increasing the concentration of BSA led to a substantial increase in the transfer of digoxin to the fetal compartment. Steady state levels of digoxin in the fetal compartment were 0.61 +/- 0.19 ng/ml at 3 mg/ml of BSA. CONCLUSION: Maternal and fetal serum concentration of BSA affect digoxin transfer in isolated perfused human placentas. Three mg/ml are considered to be the optimal albumin concentration.

Increased production of tumor necrosis factor-alpha TNF-alpha by IUGR human placentae.

OBJECTIVE: To evaluate the effect of pathological placental conditions such as intrauterine growth restriction (IUGR) or exposure to angiotensin II (AII) on TNF-alpha secretion in the vasculature of isolated human placental cotyledons. STUDY DESIGN: Isolated placental cotyledons from 10 normal and four intrauterine growth restricted fetuses were dually perfused. Perfusate samples from the fetal circulation were collected every 30 min during 120 min. TNF-alpha levels in the fetal-placental perfusate were evaluated using specific commercial ELISA kits. In three additional normal placentae, bolus injections of angiotensin II (10(-9)-10(-4) mol/l) were given into the fetal-placental circulation and perfusate samples were collected. Statistical significance of difference TNF-alpha levels between different conditions was determined by analysis of variance (ANOVA) and paired t-test. RESULTS: TNF-alpha levels were significantly higher in the perfusate of IUGR placentae as compared with normal placentae after 120 min of perfusion (mean 410+/-121 vs. 39+/-14 pg/ml, P=0.005). There was a significant dose-dependent increase in TNF-alpha levels in the placental perfusate after a bolus injection of AII 66 pg/ml with AII 10(-9) mol/l vs. 97 pg/ml with AII 10(-5) mol/l (P=0.004), respectively. CONCLUSIONS: Placental pathology related to condition IUGR might induce the secretion of proinflammatory cytokines such as TNF-alpha, which may enhance the vasoconstriction of the fetal placental vascular bed.

Neurofibromatosis in pregnancy. Maternal and perinatal outcome.

OBJECTIVE: The aim of this study was to assess the maternal and perinatal outcome in pregnant patients with neurofibromatosis (NF). STUDY DESIGN: During the period between January 1994 and December 1996 eight women with NF were delivered at the Soroka University Medical Center. Maternal age, parity, gravidy and ethnic origin were matched with a control group that included 65 healthy parturients out of a total of 31,642 deliveries that occurred in our institution during this period. Maternal outcome and perinatal complications were compared between the two groups. RESULTS: The prevalence of NF during the study period was 1:2434 deliveries. The mean gestational age at delivery was significantly lower in the study group as compared to the control group, 36.8+/-3.3 vs. 39.2+/-1.5 weeks, respectively (P=0.029). The rate of intrauterine growth restriction was significantly higher in the study group, (46.2% vs. 8.95%, respectively, P=0.0005), as well as stillbirth rate (23% vs. 1.5%, respectively, P=0.011) and cesarean section rate (38.5% vs. 7.7%, respectively, P=0.01). CONCLUSION: Patients with NF have an increased risk of perinatal complications. Thus, close antenatal observation at high risk tertiary centers is required.

Maternal serum concentrations of CA-125 in second trimester pregnancy complicated by congenital fetal anomalies.

OBJECTIVE: The purpose of the study was to determine the value of maternal serum CA-125 concentrations in pregnancies complicated by fetal anomalies with or without hydramnios. STUDY DESIGN: The study population (n=40) consisted of the following four groups of patients: (1) 10 women with abnormal maternal serum alpha fetal protein (MSAFP) in whom no fetal anomalies were observed; (2) 10 women in whom fetal anomalies were diagnosed in addition to abnormal MSAFP; (3) 10 women with fetal anomalies accompanied by hydramnios and abnormal MSAF, and (4) 10 women had normal MSAFP and were diagnosed with hydramnios without fetal anomaly. The control group consisted of 10 patients who were matched for gestational age with normal MSAFP and normal ultrasonographic examination. In all 50 cases MSAFP and maternal serum CA-125 levels were assessed. CA-125 was measured using OC 125 monoclonal antibody (IMX CA-125, Abott Lab. IL) and a value of >20 U/ml was defined as abnormal. RESULTS: Maternal serum CA-125 levels were significantly higher in the study group than in the control group, 19.8+/-15.9 U/ml and 9.9+/-4.0 U/ml (P=0.015). The difference was even greater when patients with malformed fetuses and hydramnios were compared to those with fetal anomalies and normal amount of amniotic fluid, 32.4+/-12.7 U/ml and 7.2+/-2.1 U/ml, respectively (P=0.0005). The maternal serum CA-125 levels in patients with hydramnios but without fetal anomalies were significantly lower when compared with those of the malformed fetuses and hydramnios, 9.8+/-2.3 U/ml and 32.4+/-12.7 U/ml, respectively (P=0.002). CONCLUSION: Maternal serum CA-125 is lacking in value for screening fetal structural anomalies as a significant increase in maternal serum CA-125 levels was found only in patients with fetal anomalies accompanied by hydramnios.

Vasoconstrictive activity of meconium stained amniotic fluid in the human placental vasculature.

OBJECTIVE: The purpose of this study was to study was to determine the effect of meconium stained amniotic fluid on the vasculature of isolated perfused human placental cotyledon. STUDY DESIGN: Isolated placental cotyledons were dually perfused. Fetal perfusion pressure was used as an index of vascular resistance. Meconium stained amniotic fluid (MSAF) was collected from patients after artificial rupture of membranes in term gestation. A dilution of meconium (1:2; 1:4; 1:8; 1:16) was performed. Optical density (OD) of MSAF varied between 0 and 35.0 units/g as determined by spectrophotometry. Bolus injections of 1.0 ml of MSAF at different concentrations were injected into the fetal circulation. Heated and dialyzed MSAF after adequate dilution and evaluation of optical density were injected into fetal circulation in separate experiments. RESULTS: Analysis of variance (ANOVA) and paired t-test were used for statistical analysis. Bolus injections of MSAF into the fetal circulation resulted in a concentration-dependent increase in perfusion pressure. MSAF with the highest OD resulted in a greater change in perfusion pressure as compared to more dilute MSAF (P=0.0001). After high OD amniotic fluid injections the provoked contractions lasted longer compared to dilute MSAF (P=0.006). MSAF after dialyzation did not exhibit any vasoconstrictive effect. CONCLUSION: Meconium is a vasoconstrictive agent in the fetal-placental vasculature and has a concentration dependent effect.

Indomethacin activity in the fetal vasculature of normal and meconium exposed human placentae.

OBJECTIVE: To study the effect of indomethacin on the vasculature of isolated perfused human placental cotyledon in normal and meconium pretreated placentae. STUDY DESIGN: Isolated placental cotyledons were dually perfused and fetal perfusion pressure was used as an index of vascular resistance. Meconium-stained amniotic fluid (MSAF) was collected from patients after artificial rupture of membranes, diluted 1:2, 1:4, 1:16 and 1:32 and a spectrophotometric determination of meconium concentration in amniotic fluid was performed. Only MSAF with an optical density of 20.0 units per gram was used in this study. In five placentae, the effect of indomethacin (100 microg/ml continuous perfusion from the fetal site) on basal pressure of the fetal-placental vasculature was established. In five more placentae, the effect of indomethacin on MSAF-induced vasoconstriction was established when a bolus injections of 1 ml MSAF was made into the fetal circulation. The statistical significance of response to MSAF injection was determined by paired t-test and ANOVA repeated measurements. RESULTS: A significant vasoconstrictor response to MSAF was achieved in normal placentae. Bolus injections of MSAF into the fetal circulation resulted in a significant increase in perfusion pressure (P=0.0026). Indomethacin was capable of significantly reducing the basal perfusion pressure (P=0.03). Significant attenuation of vasoconstrictor response to MSAF occurred in the presence of indomethacin (P=0.0016). CONCLUSION: Indomethacin causes a significant reduction in basal pressure of fetal placental vasculature in the human placental circulation in vitro and is capable of attenuating the vasoconstrictory activity of MSAF. The mechanism of such activity may be explained partially by the inhibitory effect of indomethacin on the PG-mediated pathways.

Is assisted hatching beneficial in patients with recurrent implantation failures?

PURPOSE: To assess the possible role of assisted hatching in patients with recurrent implantation failure during IVF cycles. DESIGN: Prospective randomized study. SETTING: IVF unit of an academic medical center. PATIENTS: Women who underwent IVF after at least three failed IVF-ET attempts. INTERVENTIONS: Patients were prospectively randomized to undergo assisted hatching of their embryos prior to their replacement by mechanical partial zona dissection. RESULTS: The study (assisted hatching) and control groups included 104 and 103 patients, respectively. There were no significant between-group differences in patient age, cause of infertility, mean number of previous IVF trials, number of oocytes retrieved, fertilization rate, or number of embryos transferred. No difference in pregnancy rate was noted on comparison of the whole study group, to the whole control group (21% and 27%, respectively). However, when the results were re-analyzed by age groups, assisted hatching was found to be harmful in the youngest group (< 34 years), significantly decreasing pregnancy rates (15% vs 35%, p < 0.05). CONCLUSION: Repeated implantation failure alone is not an indication for assisted hatching. Although assisted hatching appears to be effective in a selected group of older patients, in younger patients it may further hamper implantation and should be avoided.