Search

International audience; Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.

PCOS is one of the most common endocrine disorders affecting women and it is characterized by a combination of hyper-androgenism, chronic anovulation, and insulin resistance. While a significant progress has recently been made in the diagnosis for PCOS, the optimal infertility treatment remains to be determined. Two inositol isomers, myo-inositol (MI) and D-chiro-inositol (DCI) have been proven to be effective in PCOS treatment, by improving insulin resistance, serum androgen levels and many features of the metabolic syndrome. However, DCI alone, mostly when it is administered at high dosage, negatively affects oocyte quality, whereas the association MI/DCI, in a combination reproducing the plasma physiological ratio (40:1), represents a promising alternative in achieving better clinical results, by counteracting PCOS at both systemic and ovary level.

A substantial body of research on mammalian gametogenesis and human reproduction has recently investigated the effect of myo-inositol (MyoIns) on oocyte and sperm cell quality, due to its possible application to medically assisted reproduction. With a growing number of both clinical and basic research papers, the meaning of several observations now needs to be interpreted under a solid and rigorous physiological framework. The 2013 Florence International Consensus Conference on Myo- and D-chiro-inositol in obstetrics and gynecology has answered a number of research questions concerning the use of the two stereoisomers in assisted reproductive technologies. Available clinical trials and studies on the physiological and pharmacological effects of these molecules have been surveyed. Specifically, the physiological involvement of MyoIns in oocyte maturation and sperm cell functions has been discussed, providing an answer to the following questions: (1) Are inositols physiologically involved in oocyte maturation? (2) Are inositols involved in the physiology of spermatozoa function? (3) Is treatment with inositols helpful within assisted reproduction technology cycles? (4) Are there any differences in clinical efficacy between MyoIns and D-chiro-inositol? The conclusions of this Conference, drawn depending on expert panel opinions and shared with all the participants, are summarized in this review paper.

BACKGROUND: The follicular microenvironment is an important determinant of oocyte development. The aim of this study was to examine whether the myo-inositol (MI) content in human follicular fluid (FF) was associated with better oocyte quality. METHODS: A total of 53 patients treated with IVF was recruited to a prospective observational study. FF and serum samples collected were divided into two groups: group A consisted of FF associated with matured and fertilized oocytes, whilst group B was from follicles with immature and unfertilized oocytes. RESULTS: Patient’s age, total ampoules of HMG used, days of stimulation, basal levels of FSH, estradiol (E 2) levels on the day of HCG, and serum MI content were not significantly different between the two groups. FF volume and its MI content were significantly higher in group A compared with group B (P < 0.05). The levels of MI in FF were positively correlated with the amount of E 2 in their corresponding FF samples and also correlated with embryo quality. CONCLUSIONS: We propose that higher concentrations of MI and E2 in human FF appear to play a role in follicular maturity and provide a marker of good quality oocytes.

Objective: To determine whether oviduct mucosal cell culture supports the continued production of oviductin, a putative embryotrophic protein. Design: Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of oviductin messenger RNA (mRNA) expression after oviduct mucosal cell culture. Setting: University-based obstetrics and gynecology department. Patient(s): Ten women undergoing laparoscopy for tubal sterilization or hysterectomy for uterine fibroids.. Intervention(s): The mucosal layer was isolated from the oviduct tissue and subjected to routine culture conditions; semiquantitative RT-PCR was performed. Main Outcome Measure(s): The relationship between duration of cell culture and expression of oviductin mRNA. Result(s): There was a significant reduction in oviductin mRNA expression after 3 days in culture, with a complete loss after 6 days in 70% of the samples and after 12 days in the remaining 30%. Conclusion(s): This is the first study to investigate whether oviductin mRNA continues to be expressed in cultured human oviduct mucosal cells. Our results suggest that oviduct mucosal cells lose their ability to produce oviductin after short-term culture. This method of culture does not appear to be appropriate for a coculture system reliant upon oviductal secretion of oviductin.

Since homozygosity of the α-thalassemia-1 of Southeast Asian (SEA) type deletion results in hydrops fetalis, a novel protocol based on the real-time quantitating polymerase chain reaction (PCR) technique has been developed to quantify the intact and aberrant α-globin genes in adults. The ratio of the normal/SEA-bearing α-globin genes was expressed in cycle threshold (CT) values. Theoretically, a relative ratio of one to one was anticipated in individuals carrying the SEA type deletion. Twenty-five heterozygous and 20 normal cases were analyzed retrospectively with this protocol. Data showed that the CT values for the intact α-globin gene allele and the allele bearing the SEA type deletion in carriers were 28.74±1.49 and 26.46±2.05, respectively. Therefore, the ratio of normal/SEA type deletion-bearing α-globin genes in the carriers was 1.09±0.043. No ambiguous results were observed from other less common genotypes associated with α-thalassemia, such as the Philippine type deletion. Based on the results, we concluded that this protocol could provide a rapid method to mass screen carriers with α-thalassemia-1 of SEA type deletion in this region.

Objective: To examine changes in oviductin mRNA expression in oviductal mucosal tissue from fertile women throughout an ovulatory cycle. Design: Semiquantitative reverse-transcriptase–polymerase chain reaction (RT-PCR) analysis of oviductin mRNA. Setting: University-based obstetrics and gynecology department. Subject(s): Twenty women undergoing laparoscopy for tubal sterilization or hysterectomy for uterine fibroids. Intervention(s): The mucosal layer was isolated from the oviduct tissue, and semiquantitative RT-PCR was performed. Main Outcome Measure(s): The relationship between serum estradiol, luteinizing hormone, and progesterone concentrations and the expression of oviductin mRNA. Result(s): There was a significant positive correlation between serum estradiol and luteinizing hormone concentrations and oviductin mRNA expression. There was a significant inverse correlation between serum progesterone concentrations and oviductin mRNA expression. Conclusion(s): Little is known about the regulation of human oviductin. This study was the first to examine the relationship between oviductin mRNA expression and serum estradiol and luteinizing hormone and progesterone concentrations in fertile women. Estradiol and luteinizing hormone both have a stimulatory effect on oviductin mRNA in humans, however, it is difficult to determine whether the effects are independent of one another, as the luteinizing hormone surge is dependent on the estradiol increase. Progesterone shows a clear inhibitory effect on oviductin mRNA.

Purpose: To evaluate the effects of follicular fluid and platelet-activating factor on sperm motion characteristics of cryopreserved oligospermic and normospermic samples. Methods: Sperm motion characteristics were evaluated prior to cryopreservation, immediately after thawing and following incubation in human tubal fluid, follicular fluid, or 1-μM platelet-activating factor cultures. Sixteen oligospermic samples and 20 normospermic samples were examined. Sperm motion characteristics were analyzed manually according to WHO criteria (1999) and also with an automated videomicrography system. Result(s): Incubation in follicular fluid increased overall motility and the percentage of sperm with fast progressive motility in normospermic but not oligospermic samples. Incubation with platelet-activating factor increased overall motility and the percentage of sperm showing nonprogressive motility in both oligospermic and normospermic samples. Conclusion(s): The stimulatory effects of culture in follicular fluid as seen in normospermic samples do not show a significant benefit in oligospermic cryopreserved samples. Platelet-activating factor and follicular fluid increase motility via different mechanisms. Incubation of oligospermic cryopreserved sperm with PAF increases the number of motile sperm, thereby enabling easier identification of viable sperm for intracytoplasmic sperm injection in samples with severe asthenozoospermia.

α-Thalassemia has been estimated to account for over 60% of hydrops fetalis cases in Taiwan. The most common genotypic lesion found in α-thalassemia-1 cases in Taiwan is deletion of a large segment of the α-globin gene cluster, termed the Southeast Asian-type deletion (−SEA/; further referred to as SEA-type deletion). Seven chorionic villus samples (CVS) from pregnancies of couples both heterozygous for SEA-type deletion were studied. Non-radioactive Southern-blot hybridization using the dig-alkaline phosphatase detection system was developed to fulfill this purpose. The results were compared with corresponding polymerase chain reaction (PCR) data to elucidate the effectiveness of these two protocols in the diagnosis of the SEA-type deletion. The data showed that of the seven CVS, three demonstrated a distinctive band pattern, indicating their homozygous status of SEA-type deletion, whereas two showed heterozygous patterns, and the other two were free of the deletion. Homozygosity of the deletion was confirmed by Southern-blot hybridization performed on DNA samples extracted from the abortus tissue. However, two of the three cases with SEA-type deletion showed heterozygous PCR results. Maternal cell contamination could be responsible for the artifacts in the PCR results, but the influence due to the contamination is minimal in non-radioactive Southern-blot hybridization. We concluded that PCR is suitable for screening of carrier adults with SEA-type deletion, and non-radioactive Southern hybridization is ideal for early prenatal diagnosis of the SEA-type deletion. Am. J. Med. Genet. 95:332–335, 2000. © 2000 Wiley-Liss, Inc.

Diet is a critical factor for the development of both embryo and fetus, as well as maternal health. In particular, two natural molecules have been shown to exert beneficial effects on fertility, pregnancy wellness and embryo development: myo-inositol and melatonin, whose requirements increase during pregnancy. In the present review, we summarize the most important functions of melatonin and myo-inositol on male and female reproductive systems (oocyte quality and development, sperm quality), on the maintenance of a physiological pregnancy and on embryo development.

Myo-inositol is involved in several aspects of human reproduction. Elevated concentrations of myo-inositol in human follicular fluids appear to play a positive function in follicular maturity and provide a marker of good quality oocytes. Nevertheless its positive role in PCOS women is a consequence of a defect in the insulin signaling pathway (inositol-containing phosphoglycan mediators) that seems to be primarily implicated in the pathogenesis of insulin resistance. This article will review the involvement of inositol in female reproduction. After describing the biologic function of inositol and its derivatives, studies are quoted in which the role of inositol in fertility, oogenesis, and polycystic ovary syndrome are examined.

Purpose : To examine changes in the ratio of Bax and Bcl-2 mRNA expression throughout the ovulatory cycle in the ampullary region of the human oviduct. Methods : The mucosal layer was isolated from the human oviduct tissue and semiquantitative reverse-transcriptase polymerase chain-reaction (RT-PCR) analysis of mRNA of Bax and Bcl-2 was performed. Immunohistochemistry provided the cellular localization of the Bax and Bcl-2 proteins. The ratio of expression of Bax and Bcl-2 mRNA was examined in the ampullary region of the oviduct in samples obtained in the follicular, periovulatory, and luteal phases of the ovulatory cycle. Results : Bax expression was constant in the follicular and periovulatory phase but showed a significant increase in the luteal phase. The Bax protein was present in all oviduct mucosal epithelial cells and the intensity of staining increased in luteal phase samples. Bcl-2 was expressed at a relatively constant level throughout the ovulatory cycle. The Bcl-2 protein was present in some but not all mucosal epithelial cells and the proportion of positive cells remained constant throughout the ovulatory cycle. Conclusion : The proapoptotic gene Bax shows a significant increase in mRNA expression in the luteal phase of the ovulatory cycle while the expression level of the antiapoptotic gene Bcl-2 remains constant throughout the ovulatory cycle. The ratio of Bax:Bcl-2 increases significantly in the luteal phase consistent with cells undergoing apoptosis.

The ratio of the active progesterone receptor B isoform is higher in the ampullary region of the oviduct. Purpose: To examine mRNA expression of progesterone receptor isoforms AB and B in oviduct mucosal tissue during the ovulatory cycle and in the different functional regions of the human oviduct. Methods: The mucosal layer was isolated from human oviduct tissue and semi-quantitative RT-PCR for progesterone isoforms AB and B was performed. The RT-PCR results were verified by immunohistochemistry. Results: The isthmic region showed no mRNA expression of either progesterone receptor isoform while the relative ratio of the B isoform was significantly higher in the ampullary region compared to the fimbrial region. There was a significant increase in the ratio of PRB to PRAB in the ampullary region compared to the fimbrial region in all samples. Conclusions: We found an increase in the relative abundance of the progesterone receptor B isoform in the ampullary region which is the site of fertilization and early embryo cleavage. Our results indicate that progesterone responsive genes are more likely to be activated in the ampullary region of the oviduct due to the difference in PRAB to PRB ratio. Providing support for the hypothesis that progesterone may play a specific role in providing an appropriate environment for sperm capacitation, fertilization and early embryo cleavage.

BACKGROUND. This is the first published report of a prospective, randomized, controlled trial comparing a fixed, multi-dose GnRH antagonist protocol with a long GnRH agonist protocol in poor responders undergoing IVF. METHODS. Sixty-six poor responders were randomized into two groups: the study group received 0.25 mg of cetrorelix daily starting on day 6 of stimulation; the control group received 600mg of buserelin acetate daily starting in the mid-luteal phase of the preceding cycle. Both groups were given a fixed dose of recombinant FSH (300 IU daily) for stimulation. RESULTS. There were no significant differences in the cycle cancellation rates, duration of stimulation, consumption of gonadotrophins, and mean numbers of mature follicles, oocytes and embryos obtained. The implantation rates were similar, but the number of embryos transferred was significantly higher for the antagonist group (2.32 6 0.58 versus 1.50 6 0.83; P 5 0.01). The pregnancy rates were also higher in the antagonist group, but the difference was not statistically significant. CONCLUSION. A fixed multi-dose GnRH antagonist protocol is feasible for patients who are poor responders on a long agonist protocol; however, our study failed to demonstrate an overall improvement in ovarian responsiveness. Clinical outcomes may be improved by developing more flexible antagonist regimens, an approach that requires further evaluation.

Objective To report the birth of a healthy baby after transfer of blastocysts derived from frozen eggs and frozen spermatozoa. Design Case report. Setting University-based assisted reproduction center. Patient(s) A 37-year-old woman with secondary infertility of 4 years' duration. Intervention(s) Retrieved oocytes were cryopreserved in 1.5 M 1,2-propanediol (PROH) and 0.3 M sucrose by a slow freezing–rapid thawing protocol, semen cryopreservation, and insemination by intracytoplasmic sperm injection (ICSI) at 4 hours after thawing. Main Outcome Measure(s) Fertilization and embryo development to blastocyst stage, pregnancy, and outcome. Result(s) Ten of 14 frozen oocytes survived after thawing. Eight of them were fertilized by performing ICSI and three developed into 7- to 8-cell embryos on day 3. Two of these embryos developed into blastocysts on day 5 and were transferred. This resulted in a successful pregnancy and the delivery of a healthy baby boy. Conclusion(s) This case demonstrates the feasibility of inseminating the frozen-thawed human oocytes after 4 hours of in vitro culture and the zygotes derived from frozen oocytes and frozen spermatozoa can be cultured to blastocysts resulting in the live birth of a healthy baby boy.

Objective To determine whether oviduct mucosal cell culture with exogenous hCG or 17-β estradiol (E2) supports the continued production of oviductin, a putative embryotrophic protein. Design Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of oviductin mRNA expression after oviduct mucosal cell culture in the presence of hCG or 17-β E2. Setting University-based Obstetrics and Gynecology Department. Subject(s) Ten women undergoing laparoscopy for tubal sterilization or hysterectomy for uterine fibroids. Intervention(s) The mucosal layer was isolated from the oviduct tissue, subjected to routine culture conditions with the addition of various concentrations of hCG or 17-β E2 or the equivalent vehicle-only control and semiquantitative RT-PCR performed. Main outcome measure(s) The relationship between exposure to hCG or 17-β E2 and expression of oviductin mRNA by cultured oviduct mucosal cells. Result(s) There was a significant increase in oviductin mRNA expression after the addition of hCG to the culture medium but only in samples that had maintained a baseline level of oviductin expression. Addition of 17-β E2 to the culture medium had no significant effect on oviductin mRNA expression. Conclusion(s) Under standard cell culture conditions, baseline human oviductin mRNA expression is increased by the addition of hCG. This effect is likely to be a secondary or synergistic effect as exogenous hCG failed to restore oviductin mRNA expression in samples where expression was lost after culture. E2 failed to alter oviductin mRNA expression in oviduct mucosal cells cultured under these conditions.

Objective To determine whether oviduct mucosal cell culture with exogenous 17β E 2 supports the continued production of oviductin, a putative embryotrophic protein. Design Semiquantitative reverse-transcriptase polymerase chain reaction analysis of oviductin mRNA expression after oviduct mucosal cell culture in the presence of 17β E 2 . Three different culture systems were studied to investigate the response to E 2 . Setting University-based obstetrics and gynecology department. Subjects Oviduct tissue was obtained from 18 women undergoing laparoscopy for benign gynecologic conditions. Intervention(s) The mucosal layer was isolated from the oviduct tissue and exposed to three different culture systems, which contained various concentrations of 17β E 2 , or vehicle-only control. Main outcome measure(s) The relationship between exposure to 17β E 2 and expression of oviductin messenger (m)RNA by cultured oviduct mucosal cells. Result(s) There was a significant increase in oviductin mRNA expression after the addition of 17β E 2 to the culture system in which the in vivo cell-to-cell and cell-to-basement-membrane contacts of the oviduct had been maintained. Conclusion(s) Estradiol failed to alter oviductin mRNA expression in oviduct mucosal cells cultured under conditions in which the ciliated mucosal cell phenotype plus the cell-to-cell and cell-to-basement-membrane contacts of the oviduct were lost. However, with a culture system that maintained the cell architecture, E 2 initiated and significantly increased oviductin mRNA expression.

The study aim was to assess whether the incorporation of myo-inositol (MI) into culture medium could improve oocyte maturation in vitro.We performed a controlled prospective study using female ICR strain mice superovulated with pregnant mare's serum gonadotrophins. Cumulus-enclosed germinal vesicle (GV) oocytes were randomly cultured in medium with or without MI supplementation. The kinetics of GV breakdown after 4 h of incubation was significantly higher in oocytes incubated with 30 mmol/l of MI than in controls (P0.001). Accordingly, this concentration of MI was used for subsequent experiments. The proportion of metaphase II oocytes achieved after 24 h of culture, their fertilization and cleavage rates were significantly higher in the MI-treated group (P0.01, P0.05, P0.05 respectively). This group also demonstrated significant improvement in postimplantation development after transferring the 2-cell embryos to pseudopregnant mice. Confocal microscopy revealed spontaneous intracellular Ca(2+) oscillations within competent GV oocytes and treatment with MI caused an earlier onset of these Ca(2+) signals.Our results suggest that MI may affect meiotic progression of mouse GV oocytes possibly by enhancing the intracellular Ca(2+) oscillations. Supplementation of MI in culture medium may be useful for human oocyte maturation.

Gonadotrophin-releasing hormone (GnRH) analogues improve the outcome of treatment with IVF by increasing the number and quality of oocytes retrieved and by reducing cycle cancellation rates. Whilst short-acting GnRH analogues are most commonly used, depot preparations are now available that are more convenient for patient use. Some studies have reported that pregnancy rates with depot GnRH analogues are similar to those of short-acting preparations, but others have suggested that the more profound down-regulation seen with depot GnRH analogues results in inferior embryo quality. The purpose of this study was to determine whether a lower than conventional dose of a depot GnRH analogue may be more appropriate for use in ovarian stimulation prior to IVF. Sixty patients were randomized to receive either 3.75 mg (conventional dose) or 1.87 mg (low dose) triptorelin prior to ovarian stimulation for IVF. Suppression was measured using serum concentrations of LH measured 2 and 3 weeks after the administration of the GnRH analogues, the dose of gonadotrophin used and the time to resumption of menses. Mean concentrations of LH were 2.2 +/- 1.0 and 1.1 +/- 0.6 IU/l in the conventional dose group and 3.5 +/- 5.5 and 2.7 +/- 1.9 IU/l in the low dose group (P < 0.05 at 2 and 3 weeks). There were no significant differences between the doses of gonadotrophins used, the number of oocytes and embryos available and the time to resumption of menses, nor in the pregnancy rates. Although the degree of suppression as measured biochemically was more profound with the conventional dose, this did not affect the IVF outcome. The use of a lower dose therefore appears to be equally effective and could contribute to a reduction in the cost of treatment.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans, which are possibly associated with enhanced oxidative stress and altered MAPK pathways, respectively.

Historically, it has been theorized that the enhanced oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes arises as a direct consequence of an inability to maintain cellular glutathione (GSH) levels. This study alternatively hypothesizes that decreased NADPH concentration leads to impaired catalase activity which, in turn, underlies the observed oxidant susceptibility. To investigate this hypothesis, normal and G6PD-deficient erythrocytes and hemolysates were challenged with a H2O2-generating agent. The results of this study demonstrated that catalase activity was severely impaired upon H2O2 challenge in the G6PD-deficient cell while only a transient decrease was observed in normal cells. Supplementation of either normal or G6PD-deficient hemolysates with purified NADPH was found to significantly (P0.001) inhibit catalase inactivation upon oxidant challenge while addition of NADP+ had no effect. Analysis of these results demonstrated direct correlation between NADPH concentration and catalase activity (r = 0.881) and an inverse correlation between catalase activity and erythrocyte oxidant sensitivity (r = 0.906). In contrast, no correlation was found to exist between glutathione concentration (r = 0.170) and oxidant sensitivity. Analysis of NADPH/NADPt ratio in acatalasemic mouse erythrocytes demonstrated that NADPH maintenance alone was not sufficient to explain oxidant resistance, and that catalase activity was required. This study supports the hypothesis that impaired catalase activity underlies the enhanced oxidant sensitivity of G6PD-deficient erythrocytes and elucidates the importance of NADPH in the maintenance of normal catalase activity.

This study was designed to investigate whether the level of myo-inositol (MI) in human serum is critical for embryotrophic activity.The embryotrophic properties of human serum were evaluated by the development of postimplantation mouse embryos [in vitro assay of Tam et al. (Fertil Steril 48:834-839, 1987)]. An enzymatic spectrophotometric method using myo-inositol dehydrogenase was used for determination of serum MI. The level of MI detected in serum was compared with the embryotrophic properties and the pregnancy outcome. The effect of MI on the embryotrophic activity of human serum was studied by supplementing the suboptimal serum samples that were unsupportive of embryo growth with extra MI.Serum obtained from patients having successful IVF pregnancies generally supported better development of postimplantation mouse embryos and contained higher levels of inositol, particularly if the serum sample was collected during the IVF treatment cycle. Serum samples obtained from patients with aborted pregnancies, though supporting mouse embryo development, contained significantly lower concentrations of inositol. An improvement of the embryotrophic properties with exogenous inositol supplement was achieved in some but not all of the suboptimal serum samples studied.It is possible that other factors in addition to inositol are crucial in promoting better embryonic development.

Previous descriptions of hereditary high-phosphatidylcholine hemolytic anemia (HPCHA) have highlighted the association of increased erythrocyte membrane phosphatidylcholine with abnormal membrane cation permeability. We studied the function and composition of erythrocyte membranes from three individuals with HPCHA to characterize further the membrane abnormalities in this disorder. Despite significant macrocytosis, HPCHA red cells were dehydrated and showed an increased surface area to volume ratio compared to normal red cells. The passive efflux of K+ from HPCHA erythrocytes was increased fourfold at 37 degrees C. Total membrane phospholipid was increased 7-42%, largely due to excess phosphatidylcholine, which made up 35.8-37.2% of total phospholipid. Membrane cholesterol:phospholipid ratios were in the normal range. It appears that the excess phosphatidylcholine was not acquired during circulation, since plasma lipids were normal and all subpopulations of density-separated HPCHA erythrocytes were similarly abnormal. The ratio of total protein to phospholipid in white ghosts was increased, indicating that membrane protein was increased to an even greater extent than membrane lipids. No abnormal membrane proteins were identified by Coomassie or periodic acid Schiff (PAS) staining. Quantitation of the major membrane proteins indicated that the total protein excess in HPCHA membranes was due to a proportional increase in all major proteins. We conclude that HPCHA erythrocytes have excess membrane proteins and hypothesize that the changes in lipid composition and cation permeability are secondary to underlying protein abnormalities, which remain to be defined.

The embryotrophic property of patient's serum previously used for in vitro fertilization (IVF) was studied by culturing postimplantation organogenesis-stage mouse embryos in a medium containing equal parts of Dulbecco's modified Eagle's medium and the patient's serum. Results of embryo culture experiments were generally found to correlate well with the outcome of IVF. Serum of patients whose oocytes were fertilized and developed to cleavage-stage embryos scored highest for embryotrophic parameters such as morphologic score and protein content of the mouse embryo. There was no significant difference in terms of serum embryotrophic activity between patients who became pregnant after embryo transfer and those who did not. When mouse embryos were cultured in serum from cycles with poor IVF results, i.e., oocytes failed to be fertilized or fertilized oocytes failed to cleave or cleave abnormally, significantly retarded embryonic growth and a higher incidence of malformed embryos were observed. However, in two cases where IVF failed as a result of poor semen quality, the patient's serum was found to be supportive of mouse embryonic development.

To delineate further the role of superoxide dismutase (SOD) in red blood cell (RBC) oxidant defense, normal human erythrocytes were osmotically lysed and resealed in the presence of varying concentrations of exogenous SOD. This resulted in a dose-dependent increase in SOD activity in the resealed erythrocytes while maintaining nearly normal RBC hemoglobin concentration (less than 10% decrease from the control value), cell volume, and cellular deformability. Surprisingly, a five- or ninefold increase in SOD activity yielded no additional protection against superoxide-generating drugs (phenazine methosulfate or menadione sodium bisulfite). No significant differences were observed between the control and SOD-loaded RBCs in O2-driven methemoglobin formation or generation of thiobarbituric acid-reactive substances. In contrast, RBCs with elevated SOD activity pretreated with sodium azide (to block catalase activity) or 1-chloro-2,4- dinitrobenzene (to deplete reduced glutathione, GSH) showed significantly enhanced methemoglobin generation in response to superoxide generating drugs. No differential response was noted between the control, control-resealed, and SOD-loaded RBCs to oxidants other than superoxide. Based on our results and other data, we conclude that elevated SOD activity may imbalance cellular oxidant defense, resulting in enhanced oxidation due to the accelerated generation of H2O2, the product of O2- dismutation. This effect is significantly exacerbated under conditions in which H2O2 catabolism is altered.