Effects of supplementation with free radical scavengers on the survival and fertilization rates of mouse cryopreserved oocytes.

BACKGROUND: This study was conducted to investigate the effects of supplementation with free radical scavengers on the survival and fertilization rates of freeze-thawed mouse oocytes. METHODS: Superovulated oocytes with cumulus cells were cryopreserved by slow freezing in propanediol combined with a rapid thawing protocol. The cryopreservation medium was supplemented with the antioxidant enzymes superoxide dismutase (SOD) and catalase, and with the nitric oxide (NO) scavenger, haemoglobin (Hb). RESULTS: The addition of 50 IU/ml SOD showed significantly higher survival and fertilization capabilities compared with control (P < 0.01). Oocyte survival was greatly increased by concomitant addition of SOD with 10 IU/ml catalase (P < 0.01). On the other hand, the NO donor (sodium nitroprusside) inhibited survival and fertilization rates (P < 0.05). Significantly decreased survival and fertilization rates were also observed following the addition of high concentrations (10(-3) to 10(-6) nmol/l) of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In contrast, significantly better oocyte survival and fertilization rates were detected with low concentrations (10(-7) nmol/l) of L-NAME. Oocyte survival potential was significantly increased by addition of Hb (1 microg/ml, P < 0.05). Moreover, oocyte survival and fertilization rates were significantly promoted by the concomitant addition of SOD with Hb (P < 0.01). CONCLUSIONS: These results suggest that supplementation of free radical scavengers, particularly combinations of SOD with NO scavengers in freezing and thawing media, improved the post-thaw survival and fertilization rates of cryopreserved mouse oocytes.

Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro.

We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N-omega-nitro-L-arginine methyl ester, L-NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10(-7) M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10(-3) and 10(-5) M L-NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four-cell stage was observed by the addition of high concentration of SNP (10(-3) M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10(-5) and 10(-7) M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose-dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L-NAME reversed the inhibitory effect by each SNP or L-NAME treatment. Furthermore, low concentration of SNP (10(-7) M) but not high concentration of SNP (10(-3) M) significantly stimulated trophoblast outgrowth, whereas the addition of L-NAME suppressed the spreading of blastocysts in a dose-dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner.

The clinical efficacy of low-dose step-up follicle stimulating hormone administration for treatment of unexplained infertility.

The present study was designed to compare the clinical efficacy of low-dose step-up follicle stimulating hormone (FSH) administration with conventional FSH protocol (FSH was injected daily starting with a dose of 150 IU), both combined with intrauterine insemination (IUI), for the treatment of unexplained infertility. A total of 97 unexplained infertility couples was randomly assigned to one or other of the two treatment groups, either conventional FSH with IUI (48 patients) or low-dose step-up FSH with IUI (49 patients), and only the first treatment cycle was evaluated in each protocol. The difference in pregnancy rates per cycle was not statistically significant between the low-dose FSH group and the conventional group [seven of 49 (14.3%) and seven of 48 (14.6%) respectively]. A significant reduction in the incidence of ovarian hyperstimulation syndrome (OHSS) was observed in the low-dose group (8.3% versus 27.1%, P < 0.05). The incidence of moderate OHSS requiring hospitalization was reduced significantly in the low-dose group (low-dose 0% versus conventional 16.7%, P < 0.01). However, the low-dose protocol did not completely prevent multiple pregnancies. Our results suggest that the low-dose step-up FSH treatment appeared to be useful for the treatment of unexplained infertility because of the high pregnancy rates and the significant decrease in the incidence of OHSS.

Requirement of sperm-oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes.

We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida-free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5+/-0.6 and 4.3+/-0.6 for ICSI; 3.0+/-0.3 and 3.8+/-0.4 for activated and 0.2+/-0.1 and 0.6+/-0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore-puromycin-activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes.

Effects of low concentrations of nitric oxide on the zona pellucida binding ability of human spermatozoa.

OBJECTIVE: To investigate the direct effects of sodium nitroprusside, a nitric oxide donor, on the fertilizing ability of human spermatozoa in vitro. DESIGN: Prospective, controlled in vitro study. SETTING: IVF Unit, Medical College Hospital. PATIENT(S): Women undergoing conventional IVF. INTERVENTION(S): Human spermatozoa samples were incubated with a nitric oxide donor and a nitric oxide quencher, carboxy-imidazolineoxyl N-oxides. MAIN OUTCOME MEASURE(S): The capacitation and the acrosome reaction rates were determined by chlortetracycline assay. Sperm zona pellucida binding and sperm penetration into oocytes were determined using the hemizona assay and the human aged zona-free oocyte sperm penetration assay. RESULT(S): High concentrations of sodium nitroprusside (10(-3) and 10(-5) M) inhibited sperm motility and viability. In contrast, low concentrations of sodium nitroprusside (10(-7) and 10(-8) M) did not affect motility and resulted in increased capacitation (47 +/- 6% at 10(-7) M, 42 +/- 6% at 10(-8) M, and 24 +/- 4% in controls, respectively, P < 0.01). A twofold increase in the hemizona index occurred compared to the matched control. However, low levels of sodium nitroprusside treatment did not affect the acrosome reaction and human zona-free oocyte sperm penetration rates. CONCLUSION(S): Low concentrations of nitric oxide may have some physiologic role in fertilization through the enhancement of capacitation and zona pellucida binding but not by the induction of the acrosome reaction or the facilitation of penetration into oocytes.

Direct colorimetric monoclonal antibody enzyme immunoassay for estradiol-17 beta in saliva.

We developed a direct microtiter plate enzyme immunoassay to measure estradiol-17 beta in saliva. The assay has a commercially available monoclonal antibody, raised against estradiol-17 beta-6-carboxymethyloximebovine serum albumin, and a homologous horseradish peroxidase conjugate measured colorimetrically. The detection limit (equivalent to B0-3 SD) is 365 amol/well or 7.3 pmol/L when 50-microL samples are assayed. Cross-reactivity with estrone and estriol, testosterone, or progesterone is < 0.2%. Estradiol-17 beta was measured in daily samples over five natural menstrual cycles and eight cycles stimulated as a preliminary to in vitro fertilization, and the concentrations and fluctuations found agreed with previously published data. This method gives results in approximately 3 h and may be useful for fertility monitoring and management.

The chromosomal normality of unfertilized oocytes from patients with polycystic ovarian syndrome.

The present study was designed to compare the cycle characteristics of in-vitro fertilization (IVF) and the chromosomal normality of oocytes in patients with polycystic ovarian syndrome (PCOS) with those of patients with tubal factor infertility. In all, 28 cycles of 24 PCOS patients and 55 cycles of 31 patients with tubal factor infertility (control) were investigated. Although a significantly greater number of oocytes were retrieved from PCOS patients (mean +/- SD: 15.6 +/- 6.4 versus 9.0 +/- 4.0, PCOS versus control group, P < 0.05), the percentage of fertilized oocytes was significantly lower in the PCOS group compared with controls (40.1 versus 73.8%, P < 0.01). The pregnancy rate per embryo transfer did not differ between the two groups. Cytogenetic analysis was performed on 74 oocytes from PCOS patients and 73 oocytes from control patients. In the PCOS group, 10 of the 74 oocytes (13.5%) demonstrated aneuploidy, four (5.4%) oocytes were diploid and six (8.1%) oocytes were metaphase II with a prematurely condensed sperm chromosome (PCC). In the tubal infertility group, nine of the 73 (12.3%) oocytes showed aneuploidy, four (5.5%) oocytes were diploid and five (6.8%) oocytes were found to have PCC. There was no significant difference in the aneuploidy, diploidy and PCC rates between the two groups. These results suggest that the reduced fertilization observed in PCOS is not attributable to chromosomal aberrations or immaturity of oocytes recruited from patients with PCOS.

Mouse/human sequence divergence in a region with a paternal-specific methylation imprint at the human H19 locus.

We have identified a region with characteristics of a paternal-specific methylation imprint at the human H19 locus. This region, extending from -2.0 kb upstream to the start of transcription, is heavily methylated in sperm and on the paternal allele in somatic cells. This methylation was preserved during pre-implantation. Structural analysis revealed the presence of CpG islands and a large direct repeat with a 400 bp sequence reiterated several times, but no significant sequence homology to the corresponding region of the mouse H19 gene. These findings could suggest a role for secondary DNA structure in genomic imprinting across the species, and they also present a puzzling aspect of the evolution of the H19 regulatory region in human and mouse.

Involvement of protein kinases in platelet activating factor-induced acrosome reaction of human spermatozoa.

The involvement of protein kinases in platelet activating factor (PAF)-induced acrosome reaction of human spermatozoa was investigated using specific inhibitors of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). PAF (10(-9)-10(-11) M) treatment of spermatozoa enhanced the acrosome reaction in a dose-dependent manner (32 +/- 4% at 10(-9) M, 28 +/- 4% at 10(-10) M and 24 +/- 3% at 10(-11) M respectively). When spermatozoa were preincubated with PKA, PKC and PTK inhibitor (KT5720, calphostin C and genistein) for 15 min prior to addition of PAF, there was a significantly reduced acrosome reaction induced by PAF, but complete inhibition was not observed. On the other hand, combined use of three inhibitors completely inhibited PAF-induced acrosome reaction to levels of non-treated samples. These results suggest that the induction of the acrosome reaction by PAF treatment may involve the activation of PKA, PKC and PTK signalling pathways, and that interaction between these pathways may regulate complex mechanisms of PAF-induced acrosome reaction.

Plasma membrane block to polyspermy in human oocytes and preimplantation embryos.

The existence and time course of the human plasma membrane block to polyspermy were investigated by an in vitro fertilization assay using zona pellucida-free unfertilized oocytes, pronuclear oocytes and embryos. In the time course study using a high sperm concentration (10(5) spermatozoa ml-1), the number of penetrating spermatozoa at 30 and 60 min after insemination were 1.3 +/- 0.3 and 2.9 +/- 0.4, respectively. By 2 h after insemination, spermatozoa penetration reached a maximum. A lower maximum number of penetrating spermatozoa was observed at a low sperm concentration (10(4) spermatozoa ml-1), but the number of penetrating spermatozoa still reached a maximum by 2 h after insemination. A reinsemination experiment demonstrated that the number of penetrating spermatozoa was not significantly different between control and reinseminated oocytes, while sperm penetration was not observed in the oocyte beyond the two-cell stage. Furthermore, the number of binding spermatozoa decreased after fertilization and most of the four-cell stage embryos displayed no sperm binding. These results suggest that the plasma membrane block plays an important role in the prevention of polyspermy in the human oocyte, and that the plasma membrane block may involve permanent changes in the binding or fusion ability of spermatozoa in the oolemma after fertilization.

The role of superoxide dismutase in the human ovary and fallopian tube.

OBJECTIVES: To investigate the localization of manganese superoxide dismutase (Mn-SOD) and copper-zinc superoxide dismutase (Cu, Zn-SOD) in the human ovary and fallopian tube, and to examine the role of superoxide radicals and SODs in the human ovulatory process. METHODS: Using immunohistochemical methods, we studied the localization of SODs in 22 human ovaries, in 18 fallopian tubes, and in aspirated granulosa cells. We measured, by enzyme-linked immunosorbent assay, the concentrations of SODs in follicular fluid taken from 94 IVF patients. RESULTS: Mn-SOD was found in granulosa, in theca and luteal cells and in fallopian tubes. Cu, Zn-SOD was localized in theca and luteal cells. The concentration of Cu, Zn-SOD in follicular fluid in the high-progesterone group (11.3 + 4.2 ng/ml) was significantly less than in the low-progesterone group (24.5 + 19.5) (p < 0.05). CONCLUSION: Mn-SOD and Cu, Zn-SOD have different localizations and actions in human ovaries and fallopian tubes. The superoxide radical-SOD system might play an important role in ovulation and in the luteal function of the human ovary.

The use of zona-free aged unfertilized human oocytes as a predictor for successful subzonal insemination.

OBJECTIVE: To examine the reliability of the zona-free aged (1 day old) human unfertilized oocyte sperm penetration assay for assessing sperm fertilizing ability and to determine the predictive value of this assay for subsequent subzonal insemination (SUZI) outcomes. DESIGN: A total of 253 unfertilized oocytes from total fertilization failure patients and from good fertilization rate (> 70%) patients in standard IVF were inseminated with donors' spermatozoa, and penetration rates were compared in each group. Two hundred seventy-two unfertilized oocytes from total fertilization failure, poor fertilization (< 30%), and normal fertilization (> 30%) were inseminated with husbands' spermatozoa, and penetration rates were compared between the three groups. In 29 patients, the results of the zona-free assay performed in previous IVF were compared with the fertilization rates of subsequent SUZI. RESULTS: In the zona-free assay using donors' spermatozoa, there was no difference in penetration rates between the two groups (109/122, 89.3% versus 114/131, 87.0%). Penetration rates using partners' spermatozoa were positively correlated with fertilization rates in standard IVF (total fertilization failure 34/75, 45.3%; poor fertilization 56/77, 72.7%; normal fertilization 108/120, 90.0%). There was a significant difference in fertilization rates after SUZI between the patients with negative and positive penetration in zona-free assay (4/53, 7.5%, versus 54/174, 31.0%). CONCLUSION: The zona-free human oocyte assay may primarily reflect sperm fertilizing ability. This asssay also could be a reliable predictor for subsequent SUZI outcome.

A randomized prospective study of gonadotrophin with or without gonadotrophin-releasing hormone agonist for treatment of unexplained infertility.

The use of gonadotrophin-releasing hormone agonist (GnRHa) in combination with human menopausal gonadotrophin (HMG) for ovulation induction has been advocated for the treatment, particularly by in-vitro fertilization (IVF) of various types of infertility. The present study was designed to compare the clinical efficacy of HMG alone with a short protocol of GnRHa/HMG for treatment of unexplained infertility. A total of 91 couples with unexplained infertility were randomly assigned to one of two treatments; either HMG with intra-uterine insemination (IUI) (45 patients, 62 cycles) or GnRHa/HMG with IUI (46 patients, 69 cycles) treatments. Progesterone concentrations on the day of human chorionic gonadotrophin (HCG) administration were significantly higher in HMG (1.5 +/- 0.9 ng/ml) versus GnRHa/HMG (0.8 +/- 0.6 ng/ml; P < 0.05) cycles. Furthermore, GnRHa suppressed the occurrences of premature luteinization (GnRHa/HMG 5.8% and HMG 24.2% respectively). However, there were no significant differences in HMG dose requirements, plasma oestradiol concentrations or follicular development on the day of HCG administration between the two groups. Nor were any significant differences found in the pregnancy rates between the two treatment protocols (GnRHa/HMG 13.0% and HMG 11.3% respectively). Our results suggest no beneficial effect of GnRHa/HMG compared to HMG alone for the treatment of unexplained infertility, based on pregnancy rates.

Polynuclear fertilization in various stimulation protocols in in vitro fertilization.

OBJECTIVE: To investigate the effect of some clinical characteristics, especially various ovarian stimulation protocols, on the occurrence of polypronuclear fertilization. METHODS: One hundred forty-eight consecutive cycles in 121 patients were treated in our IVF program and 735 oocytes were analyzed retrospectively. RESULTS: Multipronuclear fertilization occurred in 33 of 530 fertilized oocytes, an incidence of 6.2%. No significant difference in the incidence of polyploidy was observed between the three stimulation protocols (CC/hMG, hMG, GnRHa/hMG). Number of polypronuclear embryos was found to be correlated with the fertilization rate, concentration of sperm inseminated, and maturity of the oocytes. CONCLUSIONS: The maturity of the oocytes and the sperm fertilizing capacity should be examined to reduce the incidence of polypronuclear fertilization.

Effects of platelet activating factor on human sperm function in vitro.

The direct effects of platelet activating factor (PAF) and the specific PAF receptor antagonist, CV-3988, on the fertilizing ability of human spermatozoa were investigated. PAF (10(-7)-10(-11) M) increased the human sperm penetration rates in a sperm penetration assay at all doses > 10(-11) M. In contrast, treatment of the spermatozoa with 10(-5) CV-3988 caused a significant decrease in human sperm penetration of zona-free hamster oocytes and adversely affected sperm motility after 24 h of incubation. This suppression was reversed by the addition of PAF. The acrosome reaction was also enhanced by PAF treatment of spermatozoa but this effect was not observed in calcium-free medium. While 10(-5) M CV-3988 decreased the acrosome reaction, the inhibition was also reversed by the addition of PAF. These results suggest that PAF may have a direct role in the fertilizing capacity of human spermatozoa. These findings also suggest that PAF may have a clinical application in an in-vitro fertilization programme.

[Pharmacokinetic and clinical studies of flomoxef in the perinatal period].

Pharmacokinetic and clinical studies on flomoxef (FMOX) in the perinatal period were carried out and following results were obtained 1. The pharmacokinetic parameter T1/2's of FMOX in maternal serum, umbilical cord serum and amniotic fluid in mothers after single intravenous injection of 1 g (n = 46) and 2 g (n = 34) were 1.11, 9.24, 9.24 hours and 2.54, 12.49, 12.49 hours, respectively. Cmax's and Tmax's of umbilical cord serum and amniotic fluid were 12.71, 11.77 micrograms/ml and 0.57, 3.35 hours upon single dose of 1 g i.v., and 35.17, 12.37 micrograms/ml and 0.32, 3.42 hours upon single dose of 2 g i.v., respectively. 2. Clinical usefulness were evaluated in 93 cases including were various infections in pregnancy and puerperal period. In pregnancy cases, clinical efficacy rate was 95.5% (21/22), and 100% in puerperal period. Bacteriological response rate was 84.6% (eradicated: 29, decreased: 4, unchanged: 2, replaced: 4 and unknown: 8 cases). No severe side effects nor clinical laboratory test results were observed in any cases. From above basic and clinical results, we conclude that FMOX is a useful and safe agent for various infections in pregnancy and puerperal period.

[Two cases of male infertility with pathological dilatation of ejaculatory duct].

Two cases of male infertility with pathological dilatation of ejaculatory duct are reported. In both cases, the dilatated wall of the ejaculatory duct was incised at the vermontanum with a cold knife endoscopically. After the incision, the findings of semen analysis of one case improved markedly and his wife became pregnant. In another case, semen analysis was not improved. Transurethral incision seemed to be a useful modality for the treatment of pathological dilatation of ejaculatory duct.

Effects of platelet activating factor on mouse sperm function.

Platelet activating factor (PAF) has been implicated in a variety of reproductive processes. This study was designed to investigate the effect of PAF and the specific PAF receptor antagonist, CV-3988, on capacitation and the acrosome reaction in mouse spermatozoa using an in vitro fertilization (IVF) system. When spermatozoa were preincubated for 30 min in medium containing PAF (10(-7) to 10(-11) M), a significant increase in the fertilization rate with both cumulus-free and zona-free oocytes was observed. In contrast, treatment of the spermatozoa with 10(-5) M CV-3988 caused a significant decrease in both sperm motility and fertilization rates with zona-intact and zona-free oocytes. This suppression was reversed by the addition of PAF. Furthermore, the acrosome reaction was enhanced by PAF treatment of spermatozoa in a dose-dependent manner. This stimulation of the acrosome reaction by PAF required the presence of calcium ions in the medium. While 10(-5) M CV-3988 inhibited the acrosome reaction, the inhibition was also reversed by the addition of PAF. These results suggest that PAF can stimulate not only the capacitation process but also the acrosome reaction, both of which are dependent on extracellular calcium.

[Role of oxygen radical and superoxide dismutase in ovulation in the rat].

We studied the effect of Cu,Zn-superoxide dismutase (SOD) and Mn-SOD, which are specific scavenging enzymes of the superoxide anion radical, on ovulation and examined the localization of SOD in rat ovaries. The results were as follows. 1) The number of ova in the Cu,Zn-SOD (8 mg x 4) administrated group (27.8 +/- 5.4 : p less than 0.01) was significantly reduced compared to the control group (49.0 +/- 3.3). Similarly, the number of ova in the Mn-SOD (2 mg x 2) administrated group (16.9 +/- 7.6 : p less than 0.01) was significantly reduced compared to the control (52.9 +/- 6.3). 2) In the rat ovary, Cu,Zn-SOD examined by the immunohistological method was found to be localized in the granulosa cells of mature Graafian follicles, growing follicles, primordial follicles and epithelial cells of the fallopian tubes. Mn-SOD was localized in the external theca cells of mature Graafian follicles and the corpus luteum. The activity of SOD estimated by the modified nitroblue tetrazolium method was consistent with the immunohistological localization of both SODs. We considered that oxygen radicals and SODs play an important role in rat ovulation, and Cu,ZN-SOD and Mn-SOD have a different localization and action in the rat ovary.