Salivary proteomics in bisphosphonate-related osteonecrosis of the jaw.

OBJECTIVE: The objective of this study was to identify differentially expressed salivary proteins in bisphosphonate-related osteonecrosis of the jaw (BRONJ) patients that could serve as biomarkers for BRONJ diagnosis. SUBJECTS AND METHODS: Whole saliva obtained from 20 BRONJ patients and 20 controls were pooled within groups. The samples were analyzed using iTRAQ-labeled two-dimensional liquid chromatography-tandem mass spectrometry. RESULTS: Overall, 1340 proteins were identified. Of these, biomarker candidates were selected based on P-value (<0.001), changes in protein expression (≥1.5-fold increase or decrease), and unique peptides identified (≥2). Three comparisons made between BRONJ and control patients identified 200 proteins to be differentially expressed in BRONJ patients. A majority of these proteins were predicted to have a role in drug metabolism and immunological and dermatological diseases. Of all the differentially expressed proteins, we selected metalloproteinase-9 and desmoplakin for further validation. Immunoassays confirmed increased expression of metalloproteinase-9 in individual saliva (P = 0.048) and serum samples (P = 0.05) of BRONJ patients. Desmoplakin was undetectable in saliva. However, desmoplakin levels tended to be lower in BRONJ serum than controls (P = 0.157). CONCLUSIONS: Multiple pathological reactions are involved in BRONJ development. One or more proteins identified by this study may prove to be useful biomarkers for BRONJ diagnosis. The role of metalloproteinase-9 and desmoplakin in BRONJ requires further investigation.

"Embryo glue" does not seem to improve chances of subsequent pregnancy in refractory in vitro fertilization cases.

PURPOSE: To determine if the use of Embryo glue improves implantation and pregnancy rates following embryo transfer (ET) in women who failed to conceive in three previous attempts. METHODS: A matched controlled study was performed in women undergoing IVF-ET, donor oocyte recipients and women using their own oocytes having fresh or frozen ETs. A woman having Embryo glue was matched with the very next woman not using glue within six months of age and having the same number of previous failed ETs. RESULTS: Embryo glue did not seem to improve pregnancy or implantation rates. In fact, in evaluating fresh embryo transfers there was a significantly higher live delivered pregnancy rate in the women not using Embryo glue (39.3%) vs those using the glue (14.3%). CONCLUSIONS: Embryo glue does not improve pregnancy outcome in women failing in previous IVF cycles.

Effect of method of oocyte fertilization on fertilization, pregnancy and implantation rates in women with unexplained infertility.

PURPOSE: To determine if intracytoplasmic sperm injection (ICSI) offers an advantage over conventional oocyte insemination for women undergoing in vitro fertilization (IVF) and embryo transfer for unexplained infertility. METHODS: A retrospective seven-year review of outcome following IVF with conventional insemination vs ICSI for the category of unexplained infertility. The decision on which method of insemination to use was made by the couple after hearing pros and cons with the consulting physician. RESULTS: There was no difference in failed fertilization rates. However, the live delivered pregnancy rates were significantly higher for the group using conventional oocyte fertilization methods. CONCLUSIONS: Because of increased embryologist time and therefore increased expense to the patient it makes more sense to first try conventional oocyte insemination over ICSI for unexplained infertility, especially since the former results in a significantly higher live delivery rate

Cryopreservation of blastocysts using a modification of a simplified freezing protocol with a one step removal of cryoprotectant successfully used previously to freeze 2 pronuclear or multi-cell embryos.

PURPOSE: To describe a modification of a simplified freezing protocol for the cryopreservation of blastocysts. METHODS: 1.5 M glycerol was substituted as a cryoprotectant instead of propanediol. RESULTS: There was a survival rate of 59.1% (13/22) with three live deliveries in seven transfers (42.9% per transfer). The implantation rate was 28.6% (4/14). CONCLUSIONS: This is the first description of a new technique for freezing blastocysts. A larger series is needed to determine if the good pregnancy rates will continue.

Length of time of embryo storage does not negatively influence pregnancy rates after thawing and transfer.

PURPOSE: To determine if longer storage of embryos in a cryopreserved state negatively affects the chance of successful implantation following thawing and transfer. METHODS: A retrospective cohort analysis of frozen-thawed embryos that had been donated to recipients. Four time periods were evaluated. RESULTS: No significant decrease in pregnancy or implantation rates was found in the longest freezing group (> or =6 years). In fact, if there was a trend, it was for improved pregnancy rates with longer storage. One of the successes was from embryos stored about 12 years. CONCLUSIONS: Hopefully these data and results from other IVF centers will influence those countries having a mandatory discarding policy to reconsider and lift these restrictions, especially to increase the pool of embryos available for donation.

The effect of blastomere number on embryo survival upon freezing/thawing.

PURPOSE: To determine if the blastomere number of embryos at the time of freezing is related to its quality post-thaw. METHODS: A retrospective cohort analysis of frozen/thawed embryos. Only multi-cell embryos were used for this study. If an embryo was of good quality it would either be transferred or re-frozen. RESULTS: There did not appear to be any trend for a lower percentage of good quality embryos with fewer numbers of blastomeres. CONCLUSIONS: Though 4-cell embryos have a markedly lower implantation potential upon fresh embryo transfer compared to 6-8-cell embryos, this is not reflected in their ability to survive freeze-thawing.

A comparison of efficacy of freezing embryos at the 2 pronuclear (2PN) stage vs multi-cell when using a simplified freezing protocol with one-step removal of cryoprotectant.

PURPOSE: To compare the efficacy of freezing embryos at the 2 pronuclear stage vs multi-cell stage using a simplified freezing protocol with a one-step removal of the cryoprotectant. METHODS: A retrospective analysis was performed. Survival, delivered pregnancy and implantation rates were compared in transfers of all embryos frozen at 2 pronuclear stage (2PN) or all embryos frozen at multi-cell stage. The results were further stratified and compared according to the number of high quality embryos transferred. RESULTS: In all categories despite comparing similar numbers and quality of embryos transferred there was a significantly higher survival rate of 2PN embryos. Significantly higher delivered pregnancy and implantation rates were seen with 2PN vs multi-cell embryos when there was only one or two embryos with > or = 6 blastomeres and < 25% fragmentation, and a trend for higher delivered pregnancy rates when there were three top quality embryos transferred. CONCLUSIONS: When given the option it is preferable when using this simplified freezing and thawing protocol to freeze at the 2PN stage.

Effect of the degree of fragmentation on embryo survival after freeze-thawing.

PURPOSE: To determine if the degree of fragmentation of embryos prior to freezing correlate in a negative manner with survival after thawing. METHODS: A retrospective review of frozen embryos thawed for purposes of embryo transfer was done. Survival and transferability rates were determined according to degree of fragmentation. RESULTS: The chance that an embryo with < 25% fragmentation was deemed good enough for transfer upon thawing was 63.6% compared to 52.8% for embryos > 25% (p < .05). CONCLUSIONS: Though more fragmented embryos have a lower survival rate after freeze thawing, about 50% of embryos with > 25% fragmentation will still survive the thaw and be able to be transferred.

Successful pregnancies from embryos cryopreserved more than ten years: two case reports.

PURPOSE: To determine the efficacy of using embryos cryopreserved more than ten years in a donor embryo program. METHODS: Embryos were cryopreserved using a simplified freezing protocol and then donated for anonymous use after the donor had had a successful pregnancy and was sure she did not want to conceive again. RESULTS: Two women in the donor embryo program transferred embryos that had been cryopreserved longer than ten years. Both patients delivered healthy babies. CONCLUSIONS: Embryos cryopreserved over ten years can result in successful pregnancies.

Evaluation of sperm nuclear morphology in specimens with abnormal sperm chromatin structural assays.

Two recent tests have claimed to identify the subfertile male even when other semen parameters were normal: the sperm chromatin structure assay (SCSA) and abnormal sperm nuclear morphology using much higher magnification. The present study attempted to determine if having a high (> 30%) DNA fragmentation index (DFI), thus resulting in an abnormal SCSA test, is associated with a greater likelihood of sperm with abnormal nuclei. Four males with high DFI scores (57.6%, 65.4%, 31.0%, and 35.3%) had their nuclei evaluated by a complex microscope set-up that magnifies the sperm at least 6000x. The corresponding % of normal nuclei was 0%, 20.0%, 23.7% and 40.0%. The mean and median % of normal nuclei was 20.9+/-16.43 and 21.8, respectively. More studies of similarly matched refractory in vitro fertilization cases, where males have normal DFI scores, are needed to determine if having a high DFI index is associated with a lower percentage of normal nuclei.

A comparison of in vitro fertilization outcome by culture media used for developing cleavage-stage embryos.

PURPOSE: To determine whether removing glucose and phosphate from media used for developing cleavage-stage embryos improves outcome following transfer of fresh or frozen embryos. Furthermore the study would evaluate the efficacy of adding nonessential amino acids and glutamate to media. METHODS: Embryo development was rotated on a weekly basis in human tubal fluid (HTF), versus two media relatively devoid of glucose and phosphate (e.g., P1), with one having the addition of essential amino acids and glutamate (Quinn's Advantage Medium). RESULTS: For fresh cycles, the implantation rate was significantly higher for Quinn's. There was less fragmentation with P1 and Quinn's. For frozen cycles, the viable pregnancy, implantation rates and embryo quality were higher for Quinn's and P1 than HTF. CONCLUSION: Removal of glucose and phosphate for day-2 embryos improves in vitro fertilization outcome after embryo transfer. It is not clear if adding certain non-essential amino acids and glutamate provides further improvement.

Effect of transferring frozen-thawed embryos resulting from fertilization of immature oocytes matured one day in culture prior to intracytoplasmic sperm injection (ICSI) on implantation rates.

PURPOSE: To evaluate whether it is efficacious to allow immature (metaphase I or germinal vesicle stage) oocytes to incubate one more day and then perform ICSI. METHOD: A retrospective study of frozen embryo transfers (ET) of deselected frozen embryos was performed to see if inclusion of a higher percentage of embryos derived from in vitro maturation of oocytes resulted in lower implantation rates. RESULTS: The implantation rate following transfer of frozen-thawed embryos, where embryos derived from immature oocytes constituted < or = 40% of the embryos (group 1) was similar to group 2 with > 40% (11.3% vs 9.8%). However, group 1 had a 10.2% implantation rate for viable (past first trimester) sacs vs only 4.3% for group 2. CONCLUSIONS: Pregnancies from transfer of frozen-thawed embryos derived from in vitro cultured immature oocytes are as likely to implant as the other deselected frozen-thawed embryos.

Sperm toxicity evaluated by the overnight sperm survival test.

A subnormal sperm stress test has also been associated with implantation failure despite apparently normal fertilization; however, this test is cumbersome and time-consuming. The overnight sperm survival test has been considered to possibly demonstrate lipid peroxidation abnormalities similar to the sperm stress test. The present study evaluated whether lower overnight survival scores were associated with lower pregnancy and implantation rates following in vitro fertilization-embryo transfer. The results showed no adverse effect of poor overnight survival test scores. Possibly, the overnight survival test, though similar in some respects to the sperm stress test, is not similar for properties of predicting embryo implantation defects. Corroboration that subnormal stress tests predicts implantation disorders is needed.

Cumulative probability of pregnancy following IVF with ICSI and fresh or frozen embryo transfer.

The authors studied the cumulative probability of pregnancy for up to 4 consecutive embryo transfer (ET) cycles with ICSI performed for male factor. Transfers could be either fresh or frozen. The clinical pregnancy rate (PR) for the first 4 cycles were similar [44% (61/366); 31% (44/138); 45% (14/31); 44% (4/9)]. Delivery rates were also similar. There was a lower PR on the second retrieval vs. the first retrieval (47% vs. 29%), but this may be related to most of the second retrievals occurring in the second transfer cycle (67%, 31/55); this may be explained by women who were poor responders and required another retrieval without a frozen ET. The majority of transfers in cycle 1 were fresh, whereas cycles 2-4 used primarily frozen-thawed embryos. These data should be helpful for patients requiring IVF with ICSI in deciding to continue with more IVF cycles or consider other

The importance of number of blastomeres when embryos are transferred in the absence of controlled ovarian hyperstimulation.

PURPOSE: To determine if the previous findings that transferring embryos with a higher number of blastomeres results in higher pregnancy rates following fresh but not frozen embryo transfer (ET) was related to the use of controlled ovarian hyperstimulation (COH) in the former but not in the latter. METHODS: Retrospective review of pregnancy and implantation rates following fresh embryo transfer of donor egg recipient cycles (where no COH is used) vs frozen ETs during the same time period according to whether there was at least one embryo with eight blastomeres transferred or not. RESULTS: Significantly higher pregnancy rates with an 8-cell ET in donor oocyte recipient cycles but not frozen ETs. CONCLUSIONS: A less favorable uterine environment caused by the use of high dose gonadotropin is not responsible for the once again observed difference in higher pregnancy rates with higher blastomere number in fresh vs frozen ET. However, an effect of the gonadotropin releasing hormone analogue was not ruled out by this study.

Highly disparate xenogeneic skin graft tolerance induction by fetal pig thymus in thymectomized mice: Conditioning requirements and the role of coimplantation of fetal pig liver.

BACKGROUND: Highly disparate xenogeneic pig skin graft tolerance and efficient repopulation of mouse CD4+ T cells are achieved in thymectomized (ATX) B6 mice that receive T cell and natural killer (NK) cell depletion by injection of a mixture of monoclonal antibodies (mAbs) (GK1.5, 2.43, 30-H12, and PK136) on days -6, -1, +7, and +14 and 3 Gy total body irradiation (TBI) followed by implantation of fetal pig thymus/liver (FP THY/LIV) grafts on day 0. The requirements for each treatment in this model to achieve pig skin graft tolerance have not previously been defined. Therefore, we performed a series of experiments to address the role of each treatment in achieving maximal skin graft tolerance. METHODS: Peripheral mouse CD4+ T-cell repopulation and pig skin graft survival were followed in this pig-to-mouse model in which recipient B6 mice were treated with modified regimens that omitted thymectomy, 3 Gy TBI, anti-Thy1.2, and anti-NK1.1 mAbs, injection of a mixture of mAbs on day +14, or coimplantation of FP LIV, respectively. RESULTS: Prolongation but not permanent survival of donor MHC-matched pig skin grafts was observed in euthymic B6 mice that received T and NK cell depletion, 3 Gy TBI, and 7 Gy thymic irradiation and FP THY/LIV in the mediastinum, suggesting that full xenogeneic tolerance was not achieved in euthymic mice. However, after grafting FP THY alone to ATX B6 mice treated either with the "standard" regimen, or with a conditioning regimen that omitted all components of the conditioning regimen except treatment with anti-CD4 and anti-CD8 mAbs, efficient peripheral repopulation of mouse CD4+ T cells and long-term donor MHC-matched pig skin graft acceptance were observed. CONCLUSIONS: Highly disparate xenogeneic pig skin graft tolerance can be achieved by grafting FP THY alone in anti-CD4 and anti-CD8 mAb-treated ATX B6 mice, but not in euthymic B6 mice. Additional treatment of ATX recipient mice with anti-Thy1.2 and NK1.1 mAbs and 3 Gy TBI is not essential for donor pig skin graft tolerance induction.

Tolerization of Gal alpha 1,3Gal-reactive B cells in pre-sensitized alpha 1,3-galactosyltransferase-deficient mice by nonmyeloablative induction of mixed chimerism.

Using a alpha 1,3-galactosyltransferase wild-type (GalT(+/+)) to deficient (GalT(-/-)) mouse bone marrow transplantation model, we have previously demonstrated that a non-myeloablative conditioning regimen is capable of permitting induction of allogeneic and xenogeneic mixed chimerism. Chimerism is associated with the rapid and lasting tolerization of anti-Gal alpha 1,3Gal (Gal) natural antibody (Ab)-producing B cells. However, one limitation of this model is that anti-Gal natural Ab levels are lower in GalT(-/-) mice than in humans and other primates. To overcome this limitation, we have now investigated the possibility of inducing such tolerance in GalT(-/-) mice that produce much higher levels of anti-Gal Abs due to presensitization with Gal-bearing xenogeneic cells. B6 GalT(-/-) mice that were pre-sensitized with rabbit red blood cells received non-myeloablative conditioning with depleting anti-CD4 and CD8 mAbs, 3Gy whole body and 7Gy thymic irradiation, and infusion of BALB/c GalT(+/+) bone marrow cells (BMC). Although engraftment of standard marrow doses was inhibited by the presensitization, long-lasting mixed chimerism could be induced in recipients of a high dose [160 x 10(6)] of allogeneic wild-type BMC. Achievement of persistent chimerism was associated with high levels of anti-Gal IgG(1) pretransplant, suggesting an inhibitory effect of non-complement-fixing IgG(1) Ab on anti-Gal-mediated marrow rejection. Induction of mixed chimerism was associated with a rapid disappearance of serum anti-Gal and tolerization of anti-Gal Ab-producing cells. B cells with anti-Gal receptors became undetectable in mixed chimeras. Mixed chimeras accepted subsequently transplanted donor-type GalT(+/+) hearts (> 140 days), whereas rapid (within 2 days) rejection of GalT(+/+) hearts occurred in conditioned control GalT(-/-) mice. In conclusion, when a high dose of GalT(+/+) BMC was administered to pre-sensitized GalT(-/-) mice, chimerism and tolerance were achieved. The absence of B cells with receptors recognizing Gal in mixed chimeras suggests a role for clonal deletion/receptor editing in the maintenance of B cell tolerance.

Pregnancy/implantation rates as related to age following transfer of frozen embryos produced by ICSI.

A study has suggested that one drawback of ICSI is that if these embryos are cryopreserved they have lower implantation rates after thawing and transfer as compared to other frozen embryos derived from conventional oocyte insemination. Other studies have not shown such adverse effects on pregnancy rates following frozen embryo transfer (ET) of embryos formed by ICSI. The study presented here evaluated the largest number of frozen ET cycles of embryos following ICSI, which were compared to couples having frozen ET with embryos formed by conventional insemination. In women age 39 and younger, the clinical, viable, pregnancy rates and implantation rates were very similar. Similar rates were reached for the older group. These data convincingly demonstrate that fertilization by ICSI does not adversely effect the implanting capacity of frozen-thawed embryos.

T cell and B cell tolerance to GALalpha1,3GAL-expressing heart xenografts is achieved in alpha1,3-galactosyltransferase-deficient mice by nonmyeloablative induction of mixed chimerism.

BACKGROUND: We have previously demonstrated that mixed xenogeneic chimerism and donor-specific T-cell tolerance can be induced in the rat-to-mouse species combination by using a relatively nontoxic, nonmyeloablative conditioning regimen. However, natural antibodies (NAbs) against Galalpha1,3Gal (Gal) pose an additional major barrier to pig-to-human vascularized xenograft acceptance. METHODS: To determine whether the mixed chimerism approach could also overcome this humoral barrier, T cell-depleted rat (GalT+/+) bone marrow cells (BMC) were transplanted to alpha1,3-galactosyltransferase deficient (GalT-/-) mice conditioned with a nonmyeloablative regimen, consisting of transient T cell and natural killer (NK) cell depletion, 3 Gy whole body irradiation, and 7 Gy thymic irradiation. RESULTS: By giving a high dose (180x106) of rat BMC, persistent mixed chimerism could be induced in GalT-/- mice, although the level of donor-type hematopoietic repopulation declined over time. Induction of mixed chimerism was associated with a rapid disappearance of anti-Gal and anti-rat NAb in the sera. Both anti-Gal Ab-producing cells and B cells with receptors recognizing Gal were undetectable in mixed chimeras, even when the chimerism levels declined, suggesting that a very low level of chimerism could effectively maintain B-cell tolerance to Gal, probably by clonal deletion and/or receptor editing. Mixed chimeras accepted subsequently transplanted donor-type rat hearts (>100 days) without immunosuppressive therapy, whereas delayed vascular and even hyperacute rejection of rat hearts occurred in conditioned control GalT-/- mice. Cellular rejection occurred by 5-6 days in conditioned control wild-type mice. CONCLUSIONS: These findings demonstrate that induction of mixed chimerism with a nonmyeloablative regimen can prevent vascularized xenograft rejection by cellular and anti-Gal Ab-dependent pathways in GalT+/+-to-GalT-/- species combinations.

Fresh embryo transfer is more effective than frozen for donor oocyte recipients but not for donors.

BACKGROUND: Recipients of donor oocytes need to be synchronized to the donor's cycle if fresh embryos are to be transferred on the cycle of oocyte retrieval. It would be much easier to merely retrieve the oocytes from the donor, fertilize the oocytes with the recipient's male partner's spermatozoa, cryopreserve the embryos, then transfer on an oestrogen/progesterone treatment programme. METHODS: The IVF outcomes of all patients enrolled in a shared oocyte programme from January 1997 to June 1999 were reviewed. Pregnancy and implantation rates were computed and statistically analysed. RESULTS: There was a significantly higher clinical pregnancy rate for recipients who had a fresh embryo transfer compared with recipients whose first embryo transfer consisted of frozen/thawed embryos (63.4 versus 43.6%). CONCLUSIONS: Conception is more likely after fresh than frozen embryo transfer with recipients but is similar to donor conception rates. If a uterine defect, per se, even without the use of the controlled ovarian stimulation regimen, could explain the difference between fresh pregnancy and implantation rates in donors versus recipients, then these same differences would have been seen when comparing frozen transfers, but they were, in fact, similar.