Peritoneal fluid from patients with endometriosis decreases sperm binding to the zona pellucida in the hemizona assay: a preliminary report.

OBJECTIVE: To evaluate the effect of peritoneal fluid (PF) from patients with endometriosis on gamete interaction under hemizona assay (HZA) conditions. DESIGN: The HZA was used to study the effect of PF from patients with endometriosis on sperm binding to the zona pellucida using media and normal PF as controls. SETTING: The patients were collected from a university hospital infertility clinic. PATIENTS: Peritoneal fluid from 16 women being evaluated for infertility or sterilization who were found to have endometriosis at surgery was used. Three normal patients, who were being sterilized, had PF that was used as a control. RESULTS: Results suggest that there is a significant reduction in the number of tightly bound sperm to the zona surface in endometriosis specimens as reflected in the hemizona index and that this effect is directly related to the stage of the disease. CONCLUSIONS: Although the sample is small, this methodology may help to elucidate one of the mechanisms responsible for endometriosis-associated infertility.     

Vitrification of human immature oocytes before and after in vitro maturation: a review

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.     

Fertilization of human oocytes by microinjection of a single spermatozoon under the zona pellucida

The authors report a method of micromanipulation for the insertion of a single spermatozoon under the zona pellucida of human oocytes that results in a high rate of fertilization without damage to the oocyte. Spermatozoa were exposed to calcium-depleted medium containing strontium chloride for 20 to 24 hours before resuspension in medium containing calcium to induce capacitation. Single spermatozoa treated in this way were injected under the zona pellucida of morphologically mature oocytes and fertilization was confirmed by electron-microscopy. Five of seven oocytes manipulated within 9 hours of aspiration from the follicle and 3 of 12 oocytes manipulated 23 to 28 hours after recovery from the follicle fertilized. This technique has profound implications for the possible treatment of severe male infertility.     

Validation of the hemizona assay in a monkey model: influence of oocyte maturational stages

The hemizona assay (HZA) was used in the monkey model to investigate sperm binding to the zona pellucida and to evaluate binding patterns according to various stages of oocyte development. Adult cynomolgus monkeys were superovulated with human menopausal gonadotropin. The oocytes were retrieved by laparoscopy, stored in salt solution, then cut into hemizonae by micromanipulation. Metaphase II oocytes showed significantly tighter binding than prophase I oocytes (P less than 0.001). Metaphase I oocytes showed intermediate binding, significantly different from the other groups (P less than 0.01). It is concluded that: (1) This study demonstrates the feasibility of HZA as a test to evaluate sperm/zona interactions in the monkey, using standards reported for human studies. (2) Oocyte meiotic competence seems to be accompanied by an increase in zona-binding properties.     

In vitro maturation and fertilization of oocytes from unstimulated ovaries: predicting the number of immature oocytes retrieved by early follicular phase ultrasonography

OBJECTIVE: To select women who will benefit most from in vitro maturation (IVM) of oocytes treatment, this study was undertaken to examine the ability of a transvaginal ultrasonography to predict the number of immature oocytes collected from unstimulated ovaries. STUDY DESIGN: The relationship between the number of immature oocytes retrieved and the pregnancy rate was assessed in 189 IVM treatment cycles. In 96 consecutive cycles, an early follicular phase transvaginal ultrasonographic measurement of the antral follicle count (AFC), ovarian volume, and peak ovarian stromal blood flow velocity (Vmax) was performed, and the results were correlated with the number of immature oocytes. RESULTS: The clinical pregnancy rate increased significantly with the number of oocytes retrieved (P =.02) and was 26.8% (15/56) in those with >10 immature oocytes. The AFC, ovarian volume, and ovarian stromal Vmax were all predictive of the number of oocytes retrieved, but when the other factors were controlled by multiple regression analysis the AFC was the only significant predictor (P <.001). CONCLUSIONS: Pregnancy rates after IVM correlate with the number of immature oocytes retrieved. This is best predicted by an ultrasonographic assessment of the AFC.     

Time interval between FSH priming and aspiration of immature human oocytes for in-vitro maturation: a prospective randomized study

This prospective randomized controlled study was performed to examine the influence of coasting for 2 days versus 3 days following a fixed daily dose of FSH for 3 days. The outcome was 2-fold. In the first experiment (n = 50 cycles), the incidence of apoptosis in granulosa cells was compared. In the second experiment (n = 28 cycles), the rates of maturation, fertilization, cleavage, pregnancy and implantation were compared. In addition, clinical pregnancy rate per aspiration was registered. Granulosa cells were collected from follicular aspirates and pooled for each patient. The APOPTAG Detection Kit was used for staining of the granulosa cells and detection of apoptosis. Oocytes were matured in vitro for 28-30 h before intracytoplasmic sperm injection. The incidence of apoptosis in granulosa cells did not differ between granulosa cells obtained after 2 days coasting (n = 25 cycles) compared with granulosa cells obtained after 3 days coasting (n = 25 cycles) (26.2 versus 26.2%). When oocytes obtained after coasting for 2 days (n = 12 cycles) were compared with oocytes obtained after coasting for 3 days (n = 16 cycles), no significant difference was found between rates of maturation (63 versus 65%), fertilization (60 versus 68%), cleavage (86 versus 92%) or implantation [5/12; 42 versus 1/12 (8%)]. A higher clinical pregnancy rate per aspiration [5/16 (31%) versus 1/12 (8%)] was obtained after coasting for 3 days compared with coasting for 2 days. The difference was not significant. This randomized study showed no difference in apoptosis of granulosa cells and no difference in developmental competence of oocytes obtained after coasting for 3 days compared with 2 days coasting.     

Hemizona assay: Use of fresh versus salt-stored human oocytes to evaluate sperm binding potential to the zona pellucida

Salt-stored human oocytes (pH 7.0) showed sperm binding ability equal to that of fresh, living oocytes under hemizona assay (HZA) conditions.     

The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential

The authors present their initial results with the hemizona assay (HZA), which was developed to predict the fertilizing potential of spermatozoa. The HZA uses the matching halves of a human zona pellucida from a nonfertilizable and nonliving oocyte, providing an internal control on zona-to-zona variability. Maximal binding of human sperm to the hemizona usually occurred after 4 to 5 hours of coincubation. Sperm from fertile men exhibited significantly higher binding capacity to hemizonae compared with sperm from men who had fertilization failure during in vitro fertilization (IVF) treatment. The HZA index is calculated as follows: (bound sperm from subfertile male) divided by (bound sperm from fertile male) X 100. These findings demonstrate that the HZA may be a useful diagnostic tool in male infertility evaluations.     

Effects of retinoic acid on maturation of immature mouse oocytes in the presence and absence of a granulosa cell co-culture system

Purpose  

Evaluation of the all-trans retinoic acid (t-RA) effects on in vitro maturation (IVM) and in vitro fertilization (IVF) of immature mouse oocytes in the presence and absence of granulosa cell monolayer.     

Retrieval of immature oocytes followed by in vitro maturation and vitrification: a case report on a new strategy of fertility preservation in women with borderline ovarian malignancy

BACKGROUND: We report a novel fertility preservation strategy in a woman with borderline ovarian tumors involving retrieval of immature oocytes, in vitro maturation (IVM) and subsequent cryopreservation. CASE: A 43-year-old woman underwent laparotomy for cystic ovarian masses on day 18 of her menstrual cycle. A diagnosis of bilateral borderline ovarian tumors was made following histological frozen section analysis. Left salpingo-oophorectomy, right ovarian cystectomy, omentectomy and lymph node sampling were performed. All visible follicles on the surface of the removed ovary were aspirated. Four immature oocytes were retrieved and underwent IVM. Three oocytes matured after 48 h and were cryopreserved. CONCLUSION: Immature oocytes can be successfully isolated from the oophorectomy specimen regardless of the day of menstrual cycle, matured in vitro and cryopreserved, providing a possible strategy for fertility preservation in this group of women.     

Human follicular fluid protease and antiprotease activities: a suggested correlation with ability of oocytes to undergo in vitro fertilization

Plasminogen activator activity was determined in human follicular fluids (FFs) obtained during in vitro fertilization procedures. The fibrinolytic activity of plasminogen activator was significantly higher in fluids from follicles that contained oocytes that were later found to fertilize in vitro (group F) as compared with fluids from follicles that contained oocytes that failed to fertilize (NF). To assess whether this difference in overt plasminogen activator activity reflects differences in conversion of an inactive, latent plasminogen activator to the active enzyme, the ability of exogenous trypsin to enhance plasminogen activation was measured. The plasminogen-dependent hydrolysis of the chromogenic substrate S-2444 in presence of trasylol (Bayer, Leverkusen, Germany) was taken as a measure of plasminogen activator activity in these experiments. No activity was found in untreated FFs, while exposure to trypsin resulted in emergence of marked plasminogen activator activity. In addition, FFs exhibited trasylol-sensitive chromogenic activity indicative of serine-protease activity. Both the plasminogen activator and serine-protease levels after tryptic activation were significantly higher in NF than in F samples. Thus, while F samples have most of their plasminogen activator in an active form, NF samples have most of their plasminogen activator in a latent, trypsin-activatable form. Follicular fluids also contain inhibitory activities toward plasmin and trypsin. The inhibition of these enzymes correlates positively with the latency of plasminogen activator. These results suggest a direct relationship between the ability of oocytes to fertilize and the overt to latent plasminogen activator activity ratios in the FFs.     

Human charcoal-stripped serum supplementation enhances both the stearoyl-coenzyme a desaturase 1 activity of cumulus cells and the in vitro maturation of oocytes

Obtaining a better outcome in assisted reproductive technology remains to be attained. In the case of in vitro fertilization (IVF), oocyte maturity is paramount for achieving a successful pregnancy. Maternal serum supplementation of in vitro maturation (IVM) medium can increase the rate of oocyte IVM. The aim of the present study was to compare the effect of whole and charcoal-stripped serum supplementation on IVM and the activity index of stearoyl-coenzyme A desaturase 1 (SCD1) in cumulus cells enclosing the oocyte as a molecular indicator of oocyte quality. Cumulus cells and germinal vesicle immature oocytes were collected from 76 women with polycystic ovarian syndrome during an IVF cycle. Serum samples were pooled from healthy women and were applied as whole or charcoal-stripped serum supplements. SCD1 expression and activity were measured by quantitative polymerase chain reaction (PCR) and gas-liquid chromatography, respectively. Charcoal-stripped serum at an amount of 10% showed a higher potency in increasing the SCD1 expression and activity index than whole serum (>1.5 fold, p?p?=?0.031). Therefore, charcoal-based lipid depletion as a simple and preparative strategy may increase the beneficial effect of serum supplementation in oocyte IVM culture.     

Influence of the dominant follicle on in-vitro maturation of human oocytes: a prospective non-randomized study

The present study was designed to examine the influence of timing of aspiration and the influence of a dominant follicle on maturation and fertility potential of immature oocytes aspirated in unstimulated cycles. The study included 81 regularly cycling women. In group I (n = 53), oocyte retrieval was scheduled the day after a follicle of 10 mm and an endometrium of at least 5 mm were observed. In group II (n = 28), aspiration was scheduled the day after observation of the same ultrasound criteria plus a detected increase (100%) in the level of oestradiol compared with the level on day 3. The maturation rate was significantly higher in group I compared with group II (107/184, 58.2% versus 56/124, 45.2%, P < 0.05), whereas the rates of fertilization and cleavage did not differ between the two groups. The clinical pregnancy rate per aspiration was significantly higher in group I compared with group II (9/53, 17% versus 0/28, 0%, P < 0.05). When comparing oocytes originating from the ovary with the dominant follicle (ipsilateral ovary) with oocytes originating from the ovary without a dominant follicle (contralateral ovary) an increased fertilization rate was observed in group I, and an increased maturation rate was observed in group II. When the data from the two groups were pooled, an increased maturation rate was observed in oocytes originating from the ipsilateral ovary compared with oocytes originating from the contralateral ovary. No difference was found with respect to rates of fertilization and cleavage rates when all oocytes originating from the ipsilateral ovary were compared with all oocytes originating from the contralateral ovary.     

Identification of macrophages at the site of peritoneal injury: evidence supporting a direct role for peritoneal macrophages in healing injured peritoneum

OBJECTIVE: To determine if peritoneal macrophages are present at the site of a surgical injury to the peritoneum during wound healing. DESIGN: Controlled research study. SETTING: Academic research laboratory. EXPERIMENTAL MODEL: A murine model of peritoneal wound healing. INTERVENTION(s): Intraperitoneal injection of polystyrene beads 1 hour after a surgical peritoneal injury to identify peritoneal macrophages. MAIN OUTCOME MEASURE(s): Presence of peritoneal macrophages at the site of the healing wound as determined by intracellular polystyrene beads on transmission electron microscopy 1, 3, 7, and 14 days after injury. RESULT(s): Peritoneal macrophages were easily distinguished from other cell types by the phagocytosis of polystyrene beads. One day after injury, peritoneal macrophages were adherent to the wound surface. By 3 days, mesothelial cells began covering the peritoneal macrophages at the wound surface and peritoneal macrophages were identified deep within the wound. Seven days after injury, the mesothelial layer was completely reconstituted, but peritoneal macrophages persisted within the healing would below the surface mesothelium. CONCLUSION(s): These data indicate that peritoneal macrophages are present at the peritoneal injury site throughout the healing interval and are consistent with these macrophages having a critical role in peritoneal wound healing.     

Obstetric variables predicting survival of the immature newborn (less than or equal to 1000 gm): a five-year experience at a single perinatal center

The immature neonate constitutes less than 3% of total births and yet accounts for almost 50% of all perinatal deaths. In a 5-year period, 476 consecutive live and inborn neonates weighing less than or equal to 1000 gm were studied. The purpose of this study was to describe our experience with these pregnancies and determine the obstetric predictors of survival. Statistical methods of univariate and multivariate analysis were used. Survival was defined as the discharge home of an alive infant. The overall survival rate without exclusions was 40.3%. The following variables were most significant and accurately predicted survivors in 76.2% and nonsurvivors in 69.2% of cases: a combination of birth weight, 5-minute Apgar score, gestational age, cervical dilatation on admission, sex, a more recent study time interval, and race. Of the factors studied, the following were directly related to advancing gestational age and birth weight: higher Apgar scores at 1 and 5 minutes, increased operative delivery rate, and increased frequency of tocolysis and glucocorticoid usage; of these factors, only the 5-minute Apgar score remained statistically significant, when controlling for gestational age and birth weight by multivariate analysis.     

Building a better mouse embryo assay: effects of mouse strain and in vitro maturation on sensitivity to contaminants of the culture environment

Purpose

The aim of this study is to compare the sensitivity of the standard one-cell mouse embryo assay (MEA) to that using in vitro-matured oocytes from hybrid and outbred mice.

Methods

The study was done by culturing embryos in the presence or absence of two concentrations (0.0005 or 0.001 %?v/v) of Triton X-100 (TX100). Embryonic development, blastocyst cell numbers (total and allocation to the trophectoderm [TE] and inner cell mass [ICM]), and blastocyst gene expression were evaluated.

Results

Neither concentration of TX100 affected (P?>?0.05) cleavage, blastocyst development, or hatching in one-cell embryos from BDF1 mice. However, all cell number endpoints were reduced (P?<?0.05) by the high concentration of TX100 and the number of ICM cells was reduced (P?<?0.05) by the low concentration of TX100. Inhibitory (P?<?0.05) effects of the high concentration of TX100 were observed in in vitro maturation (IVM) embryos from BDF1, CF1, and SW, but not ICR, mice. Cell number and allocation were negatively affected by the high concentration of TX100 in CF1 and SW embryos, but not in BDF1 or ICR embryos. The only developmental endpoints affected by the low concentration of TX100 were cleavage of BDF1 oocytes, blastocyst development of SW embryos, and cell numbers (total and inner cell mass (ICM)) of SW blastocysts.

Conclusions

The sensitivity of the MEA to TX100 is improved by using embryos from in vitro-matured oocytes, using oocytes from some outbred (SW or CF1, not ICR) strains of mice, and evaluating blastocyst cell number and allocation.

     

Human sperm microinjection into hamster oocytes: a new tool for training and evaluation of the technical proficiency of intracytoplasmic sperm injection

OBJECTIVE: To design a system for teaching intracytoplasmic sperm injection (ICSI) and to provide a standardized method to assess technical competency. SETTING: University andrology laboratory. DESIGN: Prospective study of method for training ICSI and prediction of ICSI outcome. PATIENT(S): Male infertility candidates for ICSI and fertile donors. INTERVENTION(S): Sperm from 14 fertile donors and 21 oligospermic patients were microinjected into hamster ova. Sperm head decondensation rates (SHD) and oocyte damage rates were measured. Hamster ICSI (HICSI) was used to train technicians, to assess competency, for quality control, and to predict ICSI fertilization. Sperm fertilization potential measured by HICSI was compared with the outcome of ICSI. MAIN OUTCOME MEASURE(S): Sperm head decondensation or fertilization. RESULT(S): Sperm head decondensation was observed in 425 of 773 hamster oocytes with a mean (+/- SD) rate of 60.9 +/- 15.5. Consistency was shown by repetitive testing of the same donor, comparing fresh and frozen semen, and testing of the multiple frozen aliquots of the same ejaculate. Technicians have been trained with this protocol. Excellent initial ICSI success rates for new technicians were demonstrated. Oligospermic semen samples (21 men, 251 hamster ova) tested in the HICSI test exhibited SHD rates from 12% to 100%. The poor outcome of ICSI in clinical cases was predicted by HICSI. CONCLUSION(S): The HICSI provides a method for determining the competency for the ICSI technician and interlaboratory comparison, for the prediction of success of sperm for ICSI.