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ABSTRACT: MFG-E8 is a novel endometrial protein with conserved functions in tissue remodeling and angiogenesis in non-uterine tissues. Our aims were: 1. To examine the presence of MFG-E8 protein in the human endometrium during the window of implantation, in human endometrial cell lines, in human placental tissue at different gestational ages, and in murine implantation sites during early gestation; and 2. To study the regulation of MFG-E8 mRNA expression in mice implantation sites.
MFG-E8 protein and its receptor integrin αvβ3 were detected by immunostaining in human endometrial biopsies obtained from normal volunteers, in human endometrial cell lines (epithelial: Ishikawa and HEC-1A, stromal: HESC, and endothelial: HEEC), in human products of conception from all trimesters of gestation, and in murine implantation and inter-implantation sites dissected on days 5 and 8 post-coitus. MFG-E8 gene expression was assessed by RT-PCR.
Immunohistochemical determination of MFG-E8 in endometrium and products of conception as well as relative MFG-E8 mRNA expression in mice implantation sites.
MFG-E8 protein was present almost exclusively in the epithelial compartment of human endometrium. It was also expressed in the cytotrophoblasts and syncytiotrophoblasts outlining chorionic villi of the human placenta at all trimesters of gestation, and in murine implantation sites. MFG-E8 mRNA was significantly up-regulated in murine implantation sites and with increased gestational age.
MFG-E8 expression in the endometrial epithelium as well as in chorionic villi suggests its possible role in endometrial reorganization during the receptive phase and in events related to normal pregnancy in mammals.
Placenta 07/2012; 33(10):795-802. · 3.69 Impact Factor
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S Oehninger
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ABSTRACT: The advent of in vitro fertilization and its augmentation with intracytoplasmic sperm injection (ICSI) has allowed a large number of couples suffering from moderate to severe male infertility, and also presenting with female pathologies, to achieve their reproductive dreams. Notwithstanding the existence of fundamental questions about the pathophysiological mechanisms leading to sperm dysfunction, and still unanswered concerns about health risks following ICSI, it appears that overall ICSI is safe and here to stay. Although on one hand ICSI possibly hampered advances of the knowledge in some areas of gamete biology and interaction, on the other it definitely gave impulse to studies designed to unveil the sperm contributions during and beyond fertilization, including the normalcy of the DNA/chromatin as well as molecular mechanisms of genetic/epigenetic control and nuclear organization status. In all, almost entering the fourth decade of assisted reproductive technologies, we should continue monitoring the safety of the technique and long-term development of offspring, whereas at the same time prioritizing areas of research addressing these fundamental questions.
International Journal of Andrology 06/2011; 34(5 Pt 2):e319-29. · 3.59 Impact Factor
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ABSTRACT: Globozoospermia is an infrequent pathology in which spermatozoa lack acrosomes. Patients are considered sterile without IVF augmented with intracytoplasmic sperm injection (ICSI), as fertilization is impaired due to absence of oocyte activation. As far as is known, this is the first study to report results of a comprehensive approach to the treatment of the semen parameters, sperm DNA fragmentation, aneuploidy, transmission electron microscopy, Western blotting and immunofluorescence for detection of phospholipase C zeta (PLCzeta), as well as ICSI outcome, of an affected patient. Morphological evaluation and transmission electron microscopy revealed complete globozoospermia with significant duplicate heads and tails. Analysis for DNA damage revealed fragmentation rates of approximately 80% in semen and 15-23% in swim-up fractions. PLCzeta was not detected by immunofluorescence or Western blotting. Aneuploidy rates were within normal ranges. ICSI followed by oocyte activation with calcium ionophore resulted in high rates of fertilization, and an ongoing pregnancy was established after transfer of cryopreserved-thawed embryos.
Reproductive biomedicine online 04/2010; 20(4):559-64. · 2.04 Impact Factor
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ABSTRACT: This study assessed the influence of the age of the male partner on the outcome of oocyte donation cycles. A total of 408 couples participating in 519 consecutive anonymous oocyte donation cycles were examined. Main outcome measures were fertilization rate, embryo quality, clinical pregnancy, implantation, miscarriage and live birth rates, as well as the total reproductive potential, which estimates the outcome from fresh and cryopreserved-thawed embryo transfers. A total of 241 cycles resulted in clinical pregnancy (48.5% of transfers). The mean embryo score for transferred embryos (ESTE) was higher in cycles resulting in pregnancy (P=0.003). Semen volume (P<0.001), sperm motility (P<0.001) and fertilization rate (P=0.04) decreased significantly with advanced male age, which did not correlate with mean ESTE or implantation rate. Fertilization rate was the only predictor of ESTE (B=16.066, P=0.012), whereas inseminated/retrieved egg ratio was the only predictor of implantation rate (B=0.555, P=0.039). Pregnancy was only predicted by ESTE (Exp(B)=1.023, P<0.001), which also was the only predictor of live birth (Exp(B)=1.017, P=0.009). There was no predictor of miscarriage (47 cycles, 9.1%) identified. Although semen volume, sperm motility and fertilization rate decreased with advanced male age, embryo quality, clinical pregnancy, implantation, miscarriage and live birth rates were not affected.
Reproductive biomedicine online 03/2010; 20(6):848-56. · 2.04 Impact Factor
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Ultrasound in Obstetrics and Gynecology 09/2007; 30(4):395 - 395. · 3.01 Impact Factor
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ABSTRACT: In this prospective randomized blinded clinical trial, we examined gene expression profiles of the human endometrium during the early and mid-luteal phases of the natural cycle.
An endometrial biopsy was performed on day 16 (LH +3) or on day 21 (LH +8), followed by RNA extraction and microarray analysis using an Affymetrix HG-U95A microchip. Data analysis was carried out using pairwise multiple group comparison with the significance analysis of microarrays (SAM) software.
With a false discovery rate of 0, the analysis revealed that 107 genes were significantly and differently expressed (> or =2-fold) during the early versus the mid-luteal phase of the cycle. Forty-five of these genes have not been previously linked to endometrial receptivity. Validation of the microarray data was accomplished using semiquantitative RT-PCR. We demonstrated the presence of estrogen and progesterone response elements (ERE and PRE) by analysis of the 5'-flanking regions of a subset of differentially regulated genes.
Using a strict bioinformatics approach of microarray data, we demonstrated significant changes in candidate genes during the transition of the early to the mid-luteal phase of the human endometrium that may have functional significance for the opening and maintenance of the window of implantation.
Human Reproduction 09/2005; 20(8):2104-17. · 4.47 Impact Factor
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ABSTRACT: In some animal species, the zona pellucida protein 3 (ZP3) plays a central role during fertilization, functioning as a specific receptor for sperm and as an inducer of the acrosome reaction. On the other hand, the zona pellucida protein 2 (ZP2) acts as a secondary receptor, binding to acrosome-reacted sperm. The objective of these studies was to identify ZP2 and ZP3 domains that may be of importance for the induction of the acrosome reaction. For this purpose, we synthesized a number of ZP2 and ZP3 peptides that were either conserved among species or that were species-specific according to their respective primary structures. We identified a defined, conserved ZP3 decapeptide (ZP3-6 peptide) that bound to the surface of the acrosomal region and induced the acrosome reaction in a concentration-dependent manner in capacitated bovine sperm; this effect was significant in the nanomolar range. Pertussis toxin inhibited the ZP3-6 peptide-induced acrosome reaction but had no effect on the progesterone-induced exocytotic event. Our data are in accordance with previous studies showing that progesterone induces acrosomal exocytosis via a different pathway than ZP3 and strengthen the hypothesis that the effect of ZP3-6 peptide upon acrosomal exocytosis is G protein regulated. Despite the commonly accepted idea that glycosylation of ZP proteins is required for successful sperm-oocyte interaction, we found that acrosomal exocytosis can be induced by a synthetic ZP3 peptide that is not glycosylated. The results presented in this study may be useful for the investigation of the molecular mechanisms of sperm-egg interaction in bovine and other species.
Theriogenology 05/2005; 63(6):1682-94. · 1.96 Impact Factor
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ABSTRACT: In this study we extended earlier work to determine whether sperm respond to somatic cell apoptotic stimuli and whether apoptotic phenotypes are significant indicators of human sperm quality. We evaluated ejaculated sperm from fertile donors and subfertile patients following purification of fractions of high and low motility. In unstimulated conditions, caspase enzymatic activity was higher in motile fractions from subfertile patients than in donors, and was higher in low motility fractions from both groups. Staurosporine, but not a Fas ligand or H2O2, significantly increased caspase activity, but only in high motility fractions. Procaspase-3, -7 and -9 and low levels of active caspase-3, -7 and -9 were identified by immunoblot analysis. Apoptosis-inducing factor (AIF) was present in all samples but poly ADP-ribose polymerase-1 (PARP-1) was not detected. Phosphatidylserine translocation was significantly increased only with H2O2 treatment. In ejaculates of both subfertile and fertile men, we demonstrated the presence and activation of several proteins that are key constituents of apoptosis-related pathways in somatic cells, which may serve as markers for sperm quality.
Molecular Human Reproduction 12/2004; 10(11):825-34. · 3.85 Impact Factor
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ABSTRACT: We aimed to investigate whether sperm DNA quality may predict intrauterine insemination (IUI) outcome.
The study was designed in a prospective cohort fashion, at a tertiary centre for reproductive medicine. A total of 119 patients underwent 154 cycles of IUI. Parameters related to demography, cycle management and semen sample used for IUI were evaluated. Conventional semen parameters, morphology (strict criteria), sperm DNA fragmentation and stability [evaluated by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and acridine orange staining under both acid and acid + heat denaturing conditions respectively] were measured. The main outcome measure was clinical pregnancy, defined as ultrasonographic visualization of intrauterine gestational sac(s).
Logistic regression analyses were done on six sets of data, including all cycles combined, cycles with washed samples, first cycle of each couple, first cycle of each couple with washed samples, cycles stimulated with gonadotrophins and finally gonadotrophin-stimulated cycles with washed samples. The number of pre-ovulatory follicles on day of hCG, the age of the woman and the percentage of sperm with acid- + heat-resistant DNA were the parameters that predicted IUI outcome in most of these data subsets. For the gonadotrophin-stimulated cycles, age of the man appeared as a predictor as opposed to that of the woman; and for the cycles within this subgroup, where the semen sample was washed, sperm DNA fragmentation and age of the man were the only two parameters to predict IUI outcome. No samples with >12% of sperm having DNA fragmentation resulted in pregnancy.
The number of follicles, age of the woman/man and sperm DNA quality may predict IUI outcome.
Human Reproduction 12/2002; 17(12):3122-8. · 4.47 Impact Factor
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ABSTRACT: The study aimed to evaluate the relationship between the zona pellucida induced acrosome reaction (ZIAR) and (i) percentage normal spermatozoa as well as (ii) sperm-zona pellucida binding potential among men referred for a routine semen analysis.
Semen samples of 164 consecutive men referred to the andrology laboratory for routine semen analysis were studied. Semen samples were analyzed using the new WHO standards (strict criteria). ZIAR was recorded with a lectin conjugated Pisum sativum agglutinin microassay, while sperm-zona binding was evaluated with a standard hemizona assay (HZA).
Andrology patients were divided according to the percentage normal spermatozoa in the ejaculate, namely <4% normal forms (n = 71), 5-14%, normal forms (n = 73), and >14% normal forms (n = 20). ZIAR data of the <4%, 5-14%, and >14% groups was (9.6 +/- 0.6)%, (13.9 +/- 0.5)%, and (15.0 +/- 1.1)%, respectively. The ZIAR data of fertile control men was (26.6 +/- 1.4)% which differed significantly from the three andrology referrals groups. Likewise significant differences were recorded during the hemizona assay namely, 38.0% (<4% normal forms), 54.5% (5-1% normal forms), and 62.6% (>14% normal forms). Among the group with >14% normal forms, five cases had impaired ZIAR outcome (<15%). Three of these men had normal morphology and HZAs.
ZIAR testing should become part of the second level of male fertility investigations, i.e., sperm functional testing, since 15% of andrology referrals revealed an impaired acrosome reaction response to solubilized zona pellucida.
Journal of Assisted Reproduction and Genetics 07/2002; 19(7):329-34. · 1.84 Impact Factor
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ABSTRACT: To compare IVF outcome between two protocols for luteal phase supplementation, one beginning on day 3 after oocyte retrieval and the other beginning on day 6 after retrieval.
Prospective, randomized study.
University-based assisted reproductive technology center.
One hundred twenty-six consecutive patients undergoing IVF between January and July 2000.Intervention(s): Patients were randomized to begin luteal phase support using vaginal progesterone beginning either on day 3 after oocyte retrieval or on day 6 after oocyte retrieval.
Clinical pregnancy rates and implantation rates.
All patients randomized underwent transfer. There were no differences in age, oocytes retrieved, or embryos transferred between the two groups. Those patients receiving luteal phase support with progesterone beginning on day 6 after retrieval had a significantly lower clinical pregnancy rate per transfer compared with those beginning support on day 3 after retrieval (44.8% vs. 61.0%, respectively). This difference in pregnancy rates was greater in those patients undergoing a luteal gonadotropin releasing hormone (GnRH) agonist down-regulation protocol (47.5% vs. 71.4%, day 6 vs. day 3, respectively). Beginning support on day 6 also significantly decreased implantation rates in the GnRH agonist group (21.0% vs. 34.0%, day 6 vs. day 3, respectively).
Pregnancy rates are significantly decreased by initiating luteal-phase progesterone supplementation on day 6 after oocyte retrieval during in vitro fertilization cycles.
Fertility and Sterility 01/2002; 76(6):1140-3. · 3.56 Impact Factor
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Journal of Assisted Reproduction and Genetics 11/2001; 18(10):575-7. · 1.84 Impact Factor
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ABSTRACT: This study investigated lipid peroxidation (LPO) and membrane integrity following cryopreservation-thawing.
Infertile men (study group) and donors (control group) were examined. Purified populations of highly motile spermatozoa were cryopreserved using TEST-yolk buffer and glycerol (TYB-G) followed by quick thaw. LPO was measured by a spectrophotometric assay, with and without a ferrous ion promoter. Annexin V binding was used to assess membrane translocation of phosphatidylserine (PS).
Pre-freeze LPO was significantly higher in the study than in the control group (P = 0.03). In both groups, LPO measurements after thawing were significantly higher than the pre-freeze samples not exposed to TYB-G (P = 0.002 and P = 0.001 respectively). However, when the pre-freeze samples with TYB-G were compared with the post-thaw samples (all exposed to TYB-G), these differences were not significant. There was a significant increase in PS externalization following cryopreservation in both groups (P = 0.02 and P = 0.003 respectively). In donors, pre-freeze LPO concentrations had a significant positive correlation with thawed spermatozoa depicting PS externalization (r = 0.77, P = 0.04).
Although patients had higher basal LPO than donors, LPO did not differ between fresh and cryopreserved-thawed fractionated motile spermatozoa. Freezing-thawing was associated with translocation of PS to the external membrane leaflet.
Human Reproduction 11/2001; 16(10):2148-53. · 4.47 Impact Factor
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ABSTRACT: To examine the impact of low basal cycle day 3 serum LH levels or a high FSH:LH ratio on IVF results.
A homogeneous group of patients was analyzed as identified by normal basal cycle of follicle stimulating hormone (FSH), Luteinizing hormone (LH), and estradiol (E2) levels. High responders (high LH:FSH ratio) and low responders (high FSH or E2 levels, and women > or = 42 years of age) were excluded from analysis. Only cycles stimulated with a combination of a GnRHa (luteal suppression) and pure FSH were studied.
Patients with low basal LH levels (< 3 mIU/mL) did not differ significantly from controls in terms of response to controlled ovarian hyperstimulation but there was a clear trend toward poorer implantation and clinical pregnancy rates. On the other hand, patients with a high FSH:LH ratio (> 3) had significantly fewer mature oocytes aspirated, and lower implantation and clinical pregnancy rates than patients with gonadotropin ratio < or = 3. These negative effects were evident in the presence of normal basal FSH levels and after adequate matching of female's age and number of embryos transferred.
These studies highlight a negative impact of a basal cycle high FSH:LH ratio (and possibly low LH levels) on follicular development and oocyte quality in these patients subjected to pituitary down-regulation followed by pure FSH administration. A high FSH:LH ratio may be therefore used as an early biomarker of poor ovarian response.
Journal of Assisted Reproduction and Genetics 10/2001; 18(9):499-505. · 1.84 Impact Factor
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ABSTRACT: Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells as it stabilizes the telomeres and avoids cellular senescence. The objective of this study was to modify the polymerase chain reaction-based telomeric repeat amplification protocol to detect telomerase activity in the single cell and to characterize the activity expressed in the human oocyte through to the blastocyst stage embryo. A comparative evaluation of telomerase activity and developmental stage was conducted using discarded or donated human oocytes and embryos. Telomerase activity was detected in all developmental stages evaluated from immature oocytes through to blastocyst stage embryos. Immature oocytes and blastocysts had similar levels of telomerase activity; however, both groups had significantly (P < 0.05) higher activity than zygote through to pre-morula stage embryos. Seventy-five thawed zygotes were cultured to day 3, biopsied by removing 1-2 cells, and the biopsied embryos were cultured to blastocyst stage. There was no difference (P < 0.05) in telomerase activity between cells biopsied from embryos that reached the blastocyst stage and cells from those that arrested in growth. This study has shown that human oocytes through to blastocyst stage embryos express telomerase activity, but that the level of telomerase activity in biopsied blastomeres, of the day 3 cleavage stage embryo, is not predictive of embryonic growth potential.
Molecular Human Reproduction 10/2001; 7(10):947-55. · 3.85 Impact Factor
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S Oehninger
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ABSTRACT: Male infertility is one of the most common, identifiable causes of human reproductive failure. Although considerable progress has been made toward understanding sperm physiology and the biology of gamete interaction, still more work is needed to achieve objectivity and standardization of some of the andrological diagnostic methods used in the clinical setting. More information is needed to definitively establish which tests are more accurate predictors of sperm performance and how they correlate with pregnancy potential following in vivo and in vitro interventions. Infertile men can be successfully treated with defined urological and medical therapies or with assisted reproductive technologies (ARTs). Among the latter, intracytoplasmic sperm injection (ICSI) has become a validated means to overcome multiple sperm deficiencies. Nevertheless, it is expected that simplified and more cost-efficient therapeutic modalities will be developed as additional basic (cellular-molecular) and clinical knowledge is gained.
Seminars in Reproductive Medicine 10/2001; 19(3):231-7. · 3.80 Impact Factor
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ABSTRACT: To investigate the use of donated gametes for the production of human embryonic stem cell lines.
Basic research study.
Assisted Reproductive Technology (ART) program at an academic institution.
Consenting oocyte and sperm donors.
None.
Oocytes were aspirated from oocyte donors (n = 12) and inseminated with frozen-thawed donor (n = 2) sperm followed by culture of embryos to day 5 or 6 in sequential media. The inner cell masses of expanded blastocysts were isolated using immunosurgery and cultured for 4-11 days on irradiated primary mouse embryonic fibroblasts (PMEFs). Viable cell colonies were passed every 7-10 days onto fresh PMEFs in the presence of leukemia inhibitory factor (0.1 microg/mL) and evaluated for appropriate cell surface markers.
Immunosurgery of 40 blastocysts resulted in the culture of 18 inner cell masses, which have produced three cell lines. One of these cell lines has been shown to stain positive for alkaline phosphatase and stage-specific embryonic antigen (SSEA)-4 and negative for SSEA-1, express telomerase activity, and produce hCG when allowed to differentiate.
These findings demonstrate that the future production of human embryonic stem cell lines for therapeutic use is possible with the use of donated gametes. Many ethical issues were considered before the initiation of this study, and it was our goal to ensure that both oocyte and sperm donors understood the nature and purpose of the research before their participating in the study.
Fertility and Sterility 08/2001; 76(1):132-7. · 3.56 Impact Factor
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A Monzó,
F Kondylis,
D Lynch,
J Mayer,
E Jones,
F Nehchiri,
M Morshedi,
A Schuffner,
S Muasher,
W Gibbons, S Oehninger
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ABSTRACT: To analyze the outcome of intracytoplasmic sperm injection (ICSI) cycles in infertile couples in whom the main diagnosis of infertility was azoospermia of obstructive and nonobstructive origin.
Eighty-three consecutive ICSI cycles were carried out with retrieved testicular or epididymal spermatozoa, 60 cycles in 32 patients with obstructive azoospermia and 23 cycles in 12 patients with nonobstructive azoospermia. Fifty-four testicular biopsies (testicular sperm extraction) and 18 epididymal aspirations (microepididymal sperm aspiration) were performed.Results. Motile spermatozoa were recovered in 65 cycles (90.3%). In another 3 (4.2%), nonmotile spermatozoa were retrieved. In 4 patients (5.5%), sperm could not be recovered. In 11 cycles, frozen sperm from a previous procedure were used. A significantly lower fertilization rate (64% versus 73%, P = 0.02), clinical pregnancy rate (13% versus 47%, P <0.001), and good embryo quality rates (35% versus 56%, P = 0.009) were observed in patients with nonobstructive azoospermia. In patients with obstructive azoospermia, no significant differences were observed when the outcome was analyzed on the basis of the sperm origin (ie, from testicular sperm extraction or microepididymal sperm aspiration).
When combining testicular sperm extraction or microepididymal sperm aspiration with ICSI in patients with obstructive azoospermia, the results in terms of fertilization, implantation, and pregnancy rates were similar to those found in patients with nonazoospermic obstruction who underwent ICSI with ejaculated sperm. Patients with nonobstructive azoospermia had lower fertilization, embryo quality, and pregnancy rates than did those with obstructive azoospermia, probably because of severe defects in spermatogenesis, leading to poor gamete quality. The urologist and reproductive endocrinologist now have an excellent therapeutic option to offer men with previously intractable infertility.
Urology 08/2001; 58(1):69-75. · 2.43 Impact Factor
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S Oehninger
The Lancet 07/2001; 357(9274):2068-9. · 38.28 Impact Factor
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Human Reproduction 06/2001; 16(5):1051. · 4.47 Impact Factor
