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R Abir

Publications and source records attributed to R Abir.

At least 19 recordsLinked to original sources
PubMed2006

Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults.

The signals initiating the growth of primordial follicles are unknown. Growth factors such as neurotrophin 4/5 (NT-4/5) and brain-derived neurotrophic factor (BDNF) may play a role in this process. To investigate the expression of NT-4/5 and BDNF and their receptor tyrosine kinase B (TrkB) in the early developing follicles, we fixed and froze 12 ovarian samples from adolescents/adults and 31 ovaries from human fetuses. The fixed samples were prepared for immunohistochemical staining for NT-4/5, BDNF and the TrkB receptor. Total RNA was extracted from the frozen ovarian samples, and the expression of NT-4/5, BDNF and the TrkB receptor (full length and two truncated isoforms) was investigated by RT-PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunohistochemical staining revealed the expression of NT-4/5 and BDNF mainly in oocytes and, in a minority of samples, also in the granulosa cells (GCs); TrkB receptor was identified in oocytes and GCs. Transcripts of NT-4/5, BDNF and all forms of TrkB receptor were identified in the samples. To elucidate whether indeed NT-4/5 and BDNF are involved in growth initiation of human primordial follicles, they should be added to the culture medium.

Adolescent↗
PubMed2006

In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation.

As cancer treatment improves, more young women of reproductive age are surviving, but they suffer from infertility as a consequence of the radiation and chemotherapy. Human ovarian tissue containing immature primordial follicles has been successfully cryopreserved. The ultimate aim of this technique is to induce ovarian function by re-plantation of ovarian tissue or, further into the future, by in vitro maturation (IVM) of the oocytes derived from the cryopreserved-thawed ovarian tissue, followed by routine in vitro fertilization. IVM of primordial follicles from young cancer survivors would avoid the risk of cancer re-transmission by the ovarian grafts. The present review discusses the current achievements in IVM of female germ cells and primordial ovarian follicles and the attempts to improve their development by adding various factors to the culture medium. The established methods for the evaluation of survival and growth in culture are also discussed: follicular counts, immunocytochemical methods, transmission electron microscopy, fluorescent viability markers and endocrine assays. Although the development of IVM systems is still in its infancy, researchers need to pursue their approach step-by-step, especially with regard to factors that might be involved in the activation of the ovarian follicles or female germ cells. The final measure of success will be the ability of the in vitro matured oocytes to fertilize and produce healthy offsprings. The availability of such treatment will probably lead to its demand not only by cancer patients but by other women as well.

Animals↗
PubMed2005

Presence of NGF and its receptors in ovaries from human fetuses and adults.

The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as nerve growth factor (NGF) may play a role in this process. To investigate the expression of NGF and its receptors, p75 and TrkA, in early developing follicles (mostly primordial, primary and secondary follicles), ten ovarian samples from adolescents/adults aged 13-39 and 33 ovaries from human fetuses aged 19-33 gestational weeks (GW) were obtained and immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of NGF and its two receptors. Total RNA was extracted from the frozen ovarian samples, and the expression of NGF, TrkA and p75 was investigated by RT-PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunocytochemical staining revealed the expression of NGF in granulosa cells (GC) and oocytes; TrkA was mainly in oocytes and in GC in minority of the samples; and p75 was in some of the stroma cells from fetuses aged less than 22 GW. Transcripts of NGF and TrkA were identified by RT-PCR in all samples, while those for p75 were detected only in ovarian samples from fetuses aged less than 22 GW. To elucidate if NGF is indeed involved in growth initiation of human primordial follicles, it should be added to their culture medium. The immunocytochemical detection of p75 in some of the stroma cells and transcripts in ovarian samples of fetuses less than 22 GW may suggest its role in follicular assembly.

Adolescent↗
PubMed2004

Immunocytochemical detection and RT-PCR expression of leukaemia inhibitory factor and its receptor in human fetal and adult ovaries.

The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as leukaemia inhibitory factor (LIF) may play a role in this process. To investigate the expression of LIF and its receptor in early developing follicles, nine ovarian samples from adolescents/adults aged 13-43 years and 23 ovaries from human fetuses aged 19-33 gestational weeks were immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of LIF and its two receptor units (LIF-R and gp 130). mRNA was extracted from the frozen ovarian samples, and the expression of LIF, LIF-R and gp 130 was investigated by RT-PCR. Products were resolved by 10% polyacrylamide gel electrophoresis and image analysis. There was strong to moderate immunocytochemical staining for LIF and LIF-R in oocytes from the primordial follicular stages onwards, and very weak to moderate staining for gp 130. LIF-R was also detected in granulosa cells of primary and secondary follicles from adolescents/adults. Transcripts of LIF, LIF-R and gp 130 RNA were identified by RT-PCR in all samples. The immunocytochemical staining and mRNA expression of LIF and its receptor are consistent with the concept that LIF might be involved in growth initiation of human primordial follicles through its receptor.

Adolescent↗
PubMed2001

Morphological study of fully and partially isolated early human follicles.

OBJECTIVE: To compare the development of fully and partially isolated human follicles by using various culture systems. DESIGN: Human ovarian material was incubated with collagenase and deoxyribonuclease. Fully and partially isolated follicles (30-50 microm) were dissected and studied under light and electron microscopy. The follicles were then cultured on and within various matrices. Fully isolated follicles were also cocultured with stromal cells. SETTING: Rabin Medical Center, a major care and referral center. PATIENT(S): Women undergoing laparoscopy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Microscopy studies, follicular measurements. RESULT(S): Electron microscopy studies revealed an excess of lipid droplets in the granulosa cells of freshly isolated follicles. An increase in follicular size and granulosa cell number was observed only in the fully isolated follicles cultured within collagen gels for 24 hours. Most of the partially isolated follicles detached from the collagen gels. When cultured on collagen, extracellular matrix, and poly-L-lysine, both the fully and the partially isolated follicles deteriorated within the first 24 hours; coculture with stromal cells had no beneficial effect. CONCLUSION(S): The excess in lipid droplets in granulosa cells of isolated follicles might suggest that the isolation process does not yield completely healthy follicles. However, despite this finding, our studies show that fully isolated follicles, but not partially isolated follicles, can grow within, but not on, a culture matrix.

Adolescent↗
PubMed2001

Soluble L-selectin levels during controlled ovarian hyperstimulation.

We sought to determine whether neutrophil activation, as reflected by soluble L-selectin levels, plays a role in controlled ovarian hyperstimulation (COH) and the possible correlation between soluble L-selectin and serum sex steroid levels. The study population consisted of 14 consecutive patients undergoing our routine in vitro fertilization (IVF) long gonadotropin-releasing hormone (GnRH) analog protocol. Blood was drawn three times during the COH cycle: (1) on the day when adequate suppression was obtained (Day-S); (2) on the day of, or the day prior to, human chorionic gonadotropin (hCG) administration (Day-hCG); and (3) on the day of ovum pick-up (Day-OPU). Levels of sex steroids and plasma soluble leukocyte selectin (L-selectin) were compared among the three time points. Soluble L-selectin was measured with a commercial sandwich enzyme-linked immunosorbent assay (ELISA). The results showed significantly higher levels of soluble L-selectin on Day-OPU than on Day-S and Day-hCG, and significantly lower levels on Day-hCG than Day-S. Though no significant correlations were found between soluble L-selectin and serum estradiol or hCG levels, soluble L-selectin positively correlated with serum progesterone levels. We conclude that hCG administration leads to neutrophil activation, which correlates with the degree of luteinization. Further studies are required to elucidate the relationship between the immune system and COH. These may lead to new strategies for predicting and preventing complications of COH.

Adult↗
PubMed2000

Controlled ovarian hyperstimulation--a state of endothelial activation.

PROBLEM: To aim of the study was to investigate whether controlled ovarian hyperstimulation (COH) causes endothelial activation and whether there is a correlation between endothelial activation and serum sex-steroid levels. METHOD OF STUDY: The study population consisted of 14 consecutive patients undergoing our routine IVF long gonadotropin-releasing hormone-analog protocol. Blood was drawn three times during the COH cycle: (1) day when adequate suppression was obtained (Day-S); (2) on the day of or the day prior to hCG administration (Day-hCG); and (3) on the day of ovum pick-up (Day-OPU). The levels of sex steroids and plasma soluble endothelial (E)-selectin were compared among the time points. Soluble E-selectin was measured with a commercial sandwich enzyme-linked immunosorbent assay. RESULTS: Soluble E-selectin levels were significantly higher on Day-OPU than Day-S and Day-hCG, whereas no difference was observed between Day-hCG and Day-S. No significant correlations were found between soluble E-selectin level and patient age, number of gonadotropin ampoules used, number of oocytes retrieved, or serum estradiol, progesterone and human chorionic gonadotropin levels. CONCLUSIONS: Human chorionic gonadotropin administration leads to endothelial activation regardless of the degree of ovarian response. Further studies are required to elucidate the relationship between COH and endothelial activation. These findings may lead to new strategies for predicting and preventing complications of COH, such as severe ovarian hyperstimulation syndrome.

Adult↗
PubMed1999

Pilot study of isolated early human follicles cultured in collagen gels for 24 hours.

The human ovarian cortex contains mainly primordial and primary follicles. The ability to mature these follicles in vitro could be of great importance for infertility treatments. Fresh and frozen-thawed ovarian tissue was incubated with collagenase and DNase. Follicles with one layer or an incomplete second layer of granulosa cells were then dissected. The follicles were embedded in collagen gels and cultured with Earle's balanced salt solution, 10% fetal calf serum and 0.5 IU/ml follicle stimulating hormone. Increases in the number of granulosa cell layers and in oocyte size were observed in 40 and 38.7% of the follicles from fresh and frozen-thawed tissue respectively, during a 24 h culture period. All the growing follicles were surrounded by cellular outgrowths. Attempts to culture the follicles longer resulted in deterioration of the follicles and oocyte release. Since our study was purely morphological, further growth parameters, e.g. DNA synthesis, should be examined in the future.

Adult↗
PubMed1999

Controlled ovarian hyperstimulation: a state of neutrophil activation.

PROBLEM: To investigate if controlled ovarian hyperstimulation (COH) affects the expression of neutrophil adhesion molecules and if a correlation exists between neutrophil activation and serum sex-steroid levels. METHOD OF STUDY: The pilot study was carried out in the in vitro fertilization (IVF) unit of our department, and required no modification of our routine IVF protocol. Four patients arriving for baseline hormonal profile on day 1 of the menstrual cycle before initiation of COH (control group) and 11 patients admitted for oocyte recovery (study group) were included. Venous blood was obtained from all patients and examined for hormonal profile and neutrophil activation. The latter was performed by staining for the surface adhesion molecules beta2 integrin and L-selectin. Positive cell count and mean fluorescence intensity were determined by flow cytometry. RESULTS: While neutrophil L-selectin was significantly lower in the study group than in the control group, neutrophil beta2 integrin was nonsignificantly higher. Though no significant correlations were found between neutrophil adhesion molecules and patient age, serum estradiol level, and human chorionic gonadotropin level; neutrophil L-selectin was negatively correlated with serum progesterone levels. CONCLUSIONS: COH leads to neutrophil activation, which correlates with the degree of luteinization. Further studies are required to elucidate the relationship between the immune system and COH. These may lead to new strategies for promoting fertility and preventing complications of COH.

Adult↗
PubMed1999

The role of intravenous albumin in the prevention of severe ovarian hyperstimulation syndrome. A pilot experimental study.

OBJECTIVE: To evaluate the role of intravenous albumin in the prevention of severe ovarian hyperstimulation syndrome (OHSS). DESIGN: A pilot experimental study. MATERIAL AND METHODS: Ovarian hyperstimulation was induced in 5 rabbits using human menopausal gonadotropin/human chorionic gonadotropin, after pretreatment without (control group) or with bovine serum albumin (BSA group). RESULTS: Despite an increase in serum protein levels, the BSA group showed comparable delta increase in body weight and degree of ascites formation. CONCLUSIONS: Intravenous albumin did not prevent severe OHSS in a rabbit model despite the observed increase in serum oncotic pressure.

Animals↗
PubMed1998

Preservation of fertility in women undergoing chemotherapy: current approach and future prospects.

PURPOSE: Anticancer treatment causes ovarian failure. METHODS: Some hormones may have a protective effect on the ovary. Cryopreservation (freezing) of oocytes has had very limited success, and therefore, currently its use before chemotherapy is not a feasible option. However, cryopreservation of embryos is possible. Another solution is oocyte donation followed by in vitro fertilization (IVF). RESULTS: Ovarian cortical slices containing primordial follicles have been cryopreserved successfully. To restore fertility, cryopreserved-thawed tissue taken from cancer patients before therapy could be replanted after recovery. The possible risk of malignancy restoration could be eliminated by obtaining unilaminar follicles from cryopreserved-thawed tissue and growing them in vitro, followed by routine IVF. CONCLUSIONS: Although women who undergo chemotherapy face limited options for fertility preservation, intensive studies in cryopreservation and in vitro maturation of follicles harbor hope for brighter prospects in the future.

Age Factors↗
PubMed1997

Mechanical isolation and in vitro growth of preantral and small antral human follicles.

OBJECTIVE: To develop a procedure for isolating small human follicles and to determine their growth requirements. DESIGN: Preantral and early antral follicles were isolated manually, allocated randomly to experimental groups, and cultured for a few weeks. SETTING: Patients giving informed consent in hospitals. PATIENT(S): Women undergoing laparotomy or oophorectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicular size, E2, histology. RESULT(S): Human FSH (at a dose of 1.5 U/mL) induced antral growth of follicles, and the addition of human LH (2.5 ng/mL) to human FSH stimulated growth and antral development. Histologic studies showed that most of the early antral follicles did not contain an oocyte and already had begun to undergo atresia before culturing. Levels of E2 increased in the incubation medium as the follicles increased in size, but those levels were significantly greater when the follicles contained oocytes. CONCLUSION(S): It is possible to grow small human follicles after they have been isolated manually. To develop successfully, they require a low concentration of human LH in addition to human FSH. The rate of atresia between the preantral and early antral stages in vivo is very high; therefore, it is worthwhile to develop techniques for isolating and culturing the follicles before the antral stages.

Adult↗
PubMed1997

Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture.

Ovarian cortical tissue was obtained during gynaecological operations by biopsy or after oophorectomy from 20 women aged 25-42 years. It was placed in organ culture, either fresh or following thawing after cryopreservation, for 1-4 months. The tissue was cut in slices 0.1-0.3 mm in diameter and transferred to 12 mm inserts in 24-well culture plates. These slices were cultured for 4-21 days in either alpha minimum essential medium (alpha-MEM) or Earle's balanced salt solution with added pyruvate. Both media were supplemented with 10% human serum, insulin, gonadotrophins and antibiotics. Half of the inserts were precoated with extracellular matrix (Matrigel). Histological samples revealed that there were viable, non-atretic, primordial, primary and secondary follicles in all the cultures. Mitoses were seen in the granulosa cells of the secondary follicles. Although the proportion of atretic follicles increased during culture, non-atretic follicles were still present after 21 days. After 4-11 days the proportion of viable follicles was significantly higher when cultured in Earle's solution supplemented with pyruvate, than when cultured in MEM (77 versus 38%, P < 0.001). In cultures with extracellular matrix the proportion of viable follicles was significantly higher after 10-15 days than it was without matrix (85 versus 19%, P < 0.001). Culture after thawing frozen ovarian tissue did not affect the density or the proportion of the viable follicles. Two-thirds of follicles in cryopreserved tissue were viable after 10-15 days in culture. The results indicate that it is possible to culture human primary and primordial follicles in vitro, and follicles in cryopreserved tissue are viable.

Adult↗
PubMed1996

Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants.

Pieces of ovarian cortical tissue (0.3-2 mm in diameter) were obtained during gynaecological operations by biopsy or as a result of oophorectomy from 19 women aged 19-44 years. The tissue was frozen in a programmable freezer using one of two different cryoprotectants, either 1.5 M dimethylsulphoxide (DMSO), or a combination of 1,2-propanediol (1.5 M) and sucrose (0.1 M). After cryopreservation lasting from 24 h to 5 weeks, the ovarian pieces were thawed and studied histologically. Specimens taken before and after cryopreservation with either protectant showed no signs of tissue necrosis. Follicles at similar developmental stages were found before and after freezing. The proportions of follicles showing signs of atresia, 27% in the non-frozen tissue and 19% in the frozen-thawed tissue, were not significantly different. Oocytes, too, had the same appearance after freezing and thawing with both cryoprotectants as was seen in the specimens taken before freezing. These results suggest that cryopreservation of human ovarian tissue is feasible. However, the normality of the oocytes taken from tissue which has been frozen still needs to be established. Cryopreservation of ovarian tissue would be potentially an excellent method for storage of human oocytes once methods for their maturation in vitro have been developed.

Adult↗
PubMed1996

Teratogenic IgG from sera of women with spontaneous abortions seem to induce anomalies and yolk sac damage in rat embryos. A possible method to detect abortions of immunologic origin.

PROBLEM: Spontaneous abortions due to immunological rejection of the embryo may be avoided by immunotherapy with paternal allogeneic leukocytes but there is no appropriate method to detect and differentiate this group of aborters from other groups. METHODS: In previous studies we have demonstrated that in about two-thirds of sera from women with spontaneous abortions the IgG antibodies are responsible (alone or in combination with other factors) for the embryotoxic effects of these sera on cultured rat embryos. We presently cultured 10.5-day-old rat embryos on highly teratogenic serum ("high risk" serum that induced anomalies in more than 50% of the embryos) from women with spontaneous abortions, where the IgG fraction was exchanged with IgG from control sera and vice-versa. We studied by Transmission Electron Microscopy (TEM) the extent of yolk sac damage in comparison to the rate of embryonic anomalies. RESULTS: In cases where IgG antibodies were teratogenic, embryos cultured in control sera with IgG from "high risk" sera exhibited ultrastructural yolk sac damage as well as embryonic anomalies, and the yolk sacs cultured in "high risk" sera with control IgG were normal. In cases in which the IgG exchange did not change the rate of anomalies, as IgG was not teratogenic, yolk sacs from embryos cultured in "high risk" sera remained damaged, while yolk sacs from embryos cultured in control sera after IgG exchange stayed normal. Although no significant difference in total IgG levels was found between the groups, a higher IgG1 level in sera from women with teratogenic IgG was observed in comparison to control women's sera. The obstetrical history of the women with two or more abortions who took part in our study showed that there were more cases of unknown etiology of the abortion in the women from the "high risk" group. CONCLUSIONS: The serum and the IgG fraction from women with habitual abortions can be tested in whole embryo culture to evaluate the embryonic and yolk sac damage. On this basis it may be possible to detect the women in whom the habitual abortions result from immunological rejection.

Abortion, Habitual↗
PubMed1996

Role of reactive oxygen species in diabetes-induced embryotoxicity: studies on pre-implantation mouse embryos cultured in serum from diabetic pregnant women.

Sera from diabetic patients or sera with high levels of diabetic metabolic products, were found to affect mouse and rat blastocysts. In the present study we examined the earliest developmental stages at which human diabetic serum will be lethal to mouse pre-implantation embryos, and whether reactive oxygen species (ROS) are involved in these diabetes-induced injuries. We cultured 2-4 cell-stage embryos and blastocysts in a medium containing 30 or 50% serum obtained from pregnant women with diabetes Type I, Type II and gestational diabetes (GDM) for 72 h. The development of the 2-4 cell-stage embryos was delayed when cultured in 30% diabetic serum, but the viability was impaired to a lesser extent. Viability was reduced in blastocysts cultured in 50% diabetic serum, but the development of the living embryos was not delayed. Cyclic voltametry measures the oxidation potential of the tissue and the concentration of antioxidants, thus reflecting the total antioxidative activity of the embryos. Pre-implantation embryos cultured in diabetic serum had a lower concentration of antioxidants than embryos cultured in non-diabetic serum. It seems, therefore, that diabetic metabolic factors may induce embryotoxicity in pre-implantation embryos through derangement of the antioxidant defense mechanism. A similar mechanism is suggested for the diabetes-induced teratogenicity in post-implantation embryos.

Animals↗
PubMed1994

The effects of sera from women with spontaneous abortions on the in vitro development of early somite stage rat embryos.

PROBLEM: Spontaneous abortions occur in 40 to 50% of pregnancies, but the causes for some abortions, especially those that are recurrent (spontaneous), are still unknown. METHOD: Following previous studies that demonstrated embryotoxic effects of sera from women with spontaneous abortions in preimplantation mouse embryos, we cultured 10.5-day-old rat embryos in sera from women after spontaneous abortions to look for specific teratogenic effects. RESULTS: About 50% of the embryos cultured in sera from women after spontaneous abortions were malformed, as compared to 19.1 and 27.1% malformations in embryos cultured in sera from women after a normal delivery and during a normal second trimester of pregnancy, respectively. We divided the sera from women who had spontaneous abortions into high-risk, and low-risk sera. In the high-risk sera from one abortion, we found 74.2% malformed embryos and in the high-risk group from two or more abortions this rate was 81.0%. This is compared to a rate of 17.1 and 10.3% in the low-risk sera, respectively. We have also found lower DNA and protein synthesis in the embryos cultured in high-risk sera compared to those cultured in low-risk and control sera. Transmission electron microscopy examination of yolk sacs cultured in high risk sera showed ultrastructural damage as represented by a lower number of microvilli and a higher number of inclusions in the entodermal cells when compared to controls. Amino acid chromatography of the serum and the concentrations of folic acid and zinc were similar in control and high-risk sera. CONCLUSION: It seems that the majority of sera from women with unexplained spontaneous abortions are teratogenic to rat embryos in culture. In about two-thirds of these sera the teratogenic factor(s) seem to be present in the IgG fraction.

Abortion, Habitual↗