Pregnant Recipients Research Articles

The usefulness of a single measurement of insulin-like growth factor-1 as a predictor of embryo yield and pregnancy rates in a bovine MOET program

The objective was to determine if a single measurement of plasma insulin-like growth factor-1 (IGF-1) could predict the number of viable embryos obtained from donors and the likelihood of pregnancy in recipients in multiple ovulation and embryo transfer (MOET) programs in cattle. The embryo yields from 101 embryo recoveries were examined in maiden Holstein heifers ( n = 75) and multiparous Holstein cows (lactating cows n = 20, dry cows n = 6). Donors were superstimulated with FSH and embryo recovery was done non-surgically 7 days after artificial insemination. Embryos were classified according to the IETS criteria. Pregnancy rates in 100 maiden Holstein heifer recipients were analysed. Recipients were on day 7 ± 1 of the estrous cycle at transfer. Pregnancy diagnosis was carried out at day 30 (PD30) and rechecked at day 60 (PD60) after transfer. Blood samples from coccygeal vessels taken at the time of embryo recovery (donors) and transfer (recipients) were analysed for IGF-1, insulin, β-hydroxybutyrate (β-OHB), non-esterified fatty acids (NEFA), urea and cholesterol. There was a negative correlation between the number of viable embryos and insulin ( r = −0.33, P = 0.025) in donor heifers. In donor cows, the number of viable embryos was correlated with IGF-1 ( r = 0.43, P = 0.028) and cholesterol ( r = −0.43, P = 0.027). In recipients, PD30 and PD60 were not affected by any of the circulating parameters analysed. Insulin, IGF-1 and cholesterol only explained 8.9, 13.9 and 15.8% of the variation in the production of viable embryos, respectively. Several factors affect MOET programs and under the circumstances of the present study the usefulness of hormonal and metabolic profiles as predictors of the outcome of this biotechnology was limited.

Successful use of oral ganciclovir for the treatment of intrauterine cytomegalovirus infection in a renal allograft recipient

Congenital cytomegalovirus (CMV) infection occurs in approximately 1% of newborns and is the leading infectious cause of congenital birth defects. Female renal allograft recipients who develop CMV infection during pregnancy are at risk for both graft dysfunction and fetal morbidity. DNA-based analysis of amniotic fluid (AF) from at-risk pregnancies has been suggested as an adjunct/substitute for traditional culture. We have shown that CMV-polymerase chain reaction of AF is a useful diagnostic test for congenital CMV infection. Using this test we diagnosed CMV infection in the fetus of a 30-year-old renal transplant recipient. As termination was not an option for the family, the patient was extensively counseled and treated with oral ganciclovir. This resulted in clearance of the virus from the AF and the delivery of a healthy newborn girl, free of CMV disease. This is the first reported case to our knowledge of successful use of maternal ganciclovir to treat intrauterine CMV infection in a pregnant renal transplant recipient.

Pregnancy and Implantation Rates of Donor Oocyte Recipients Obtaining Oocytes from Infertile Donors with Unexplained Infertility are Comparable to the Rates Obtained from Oocytes from Donors Whose Infertility Etiology is Tubal or Male Factor

Background and Significance: There are two main methods for supplying oocytes to women with decreased egg reserve: paid donors and donors who are trying to get pregnant by in vitro fertilization (IVF) who share half of the oocytes retrieved for reducing the cost of their own IVF procedure. Many recipients are fearful of selecting a shared donor with unexplained infertility fearing that the oocyte may be the problem, and thus only a small percentage of chosen donors have the diagnosis of unexplained infertility. Objective: The purpose of the study was to determine if a donor oocyte recipient who selects oocytes from an infertile woman with unexplained infertility has less of a chance of conception and delivery than if the oocytes were selected from infertile donors with known tubal or male factor infertility. Materials and Methods: A retrospective review of donor oocyte recipient cycles from over six years was conducted. If the recipient elected to freeze all embryos without a fresh transfer these cycles were eliminated. If a donor had endometriosis but normal tubes and a normal male partner this was considered as unexplained infertility. Results: There were 159 shared oocyte cycles evaluated as well as 403 paid oocyte fresh embryo transfers. The fertilization rates and clinical ongoing/delivered pregnancy rates and implantation rates are seen in the table below. Though there were only 12 transfers in the endometriosis and unexplained category, there were 8 clinical pregnancies (66.7% clinical pregnancy rate/cycle) and 7 ongoing delivered pregnancies (58.3%). For this combined group there were 12 embryos that implanted out of 41 that were transferred (29.2% implantation rate). Tabled 1EndometriosisTubalMale factorUnexplainedPaid donor# transfers590517394# eggs inseminated1031369660936457% fertilized69.968.264.180.669.3Avg. # embryos transferred4.03.33.23.03.0% implanted25.035.828.433.329.8% clinical pregnancy per transfer60.064.456.971.456.6% ongoing/delivered per transfer60.060.047.157.151.0 Open table in a new tab Conclusion: Donor oocytes obtained from women with unexplained infertility/endometriosis result in comparable pregnancy and implantation rates as paid donors and shared donors with diagnosis of tubal factor and male factor. Hopefully these data will encourage more recipients to choose from this category of shared donors and a larger sample size can be evaluated in the future. Though not the focus of the study, these data also show that oocytes from shared infertile oocyte donors provide recipients with comparable outcome as paid donors.

Levonorgestrel hormone releasing intrauterine system (Mirena®) as a contraceptive in egg donors: case report

We report the safe use of levonorgestrel hormone releasing intra uterine system (Mirena) as a contraceptive in egg donors during a treatment cycle. In the first case report, a 29-year-old egg donor using the Mirena coil for contraception and two egg recipients, aged 41 years and 32 years respectively underwent standard IVF treatment, oocyte retrieval in the egg donor and in vitro fertilization followed by embryo transfer in the recipient. The outcome of IVF cycle using donor eggs was satisfactory with successful pregnancy in the egg recipient. The second case involved a 34-year-old egg donor using the Mirena coil and a 44-year-old recipient. Our findings suggest that egg donors can safely use the (Mirena) as a contraceptive device during treatment, without compromising follicular development and oocyte quality.

36 IMPROVING THE APPLICATION OF NUCLEAR TRANSFER FOR PRODUCING NON-DOMESTIC FELIDS

One of the most remarkable aspects of somatic cell nuclear transfer (NT) is the possibility of avoiding extinction when there are few remaining animals of a specific felid population. Previously, we produced live male African Wildcat (AWC; Felis lybica) cloned kittens using inter-species nuclear transfer (Gomez et al. 2004 Cloning and Stem Cells 6, 217–228). The production of females is a primary objective of most breeding programs. Therefore, the purpose of the present study was to determine (1) if we could produce live female AWC cloned kittens at a proportion similar to that previously demonstrated with males, and (2) if our inter-species NT technique used to produce AWC is applicable to in vitro production of another non-domestic felid species. Specifically, we evaluated the in vivo developmental competence of NT embryos derived by fusion of Black footed cat (BFC, Felis nigripes) and AWC fibroblasts with domestic cat (DSH, Felis catus) cytoplasts, after transfer into domestic cat recipients. Fibroblast cell lines were established from skin biopsies of BFC (6-year-old), and AWC (12-year-old) adult females. After at least three passages, cells were serum-starved for 5 days and injected into the perivitelline space of enucleated domestic cat oocytes. Fusion of cell-cytoplast couplets was induced by applying a 3-s AC pre-pulse of 20 V, 1 MHz, followed by two 30-μs DC pulses of 240 V/mm. Fused couplets were activated 2 to 3 h after fusion by exposure to two 60 μsec DC pulses of 120 V/mm, followed by 4 h incubation with 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B. Reconstructed BFC (n = 16) and AWC (n = 536) NT Day 1 embryos were transferred by laparoscopy into the oviducts of 1 and 12 gonadotrophin-treated DSH recipients, respectively, on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. One cat (100%) receiving BFC NT embryos and 5 (41.6%) cats receiving AWC NT embryos became pregnant. Twenty-three AWC cloned embryos implanted and 11 kittens were born. Three BFC NT embryos implanted and the pregnancy is currently ongoing. AWC cloned kittens were phenotypically and genetically identical to their somatic cell donor. Their clonal identity was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. In summary, these results indicate that female AWC cloned kittens can be produced and BFC pregnancy can be established in domestic cat recipients. The embryo implantation rate and viability of AWC female cloned embryos was higher than that observed after the transfer of AWC male cloned embryos. The difference may be due to improvements in the NT procedure, rather than to differences in the sex of the cell lines. Table 1. Implantation rate and fetal survival to term of AWC and BFC NT embryos in pregnant domestic cat recipients

290 THE FACTORS ON RATES OF ABNORMALITY, DISEASE AND MORTALITY OF CALVES DERIVED FROM IN VITRO EMBRYOS OF KOREAN NATIVE CATTLE

In Korea, in vitro production and transfer of bovine embryos has advanced remarkably and applied commercially. However, in vitro-produced embryos result in lower pregnancy and higher abortion rates and in some cases increased rates of abnormality and mortality in calves. The present study was conducted to investigate the effects of various factors such as recipient parity, delivery season, offspring number, pregnancy period, delivery type, midwifery type, dystocia and vaccination, on the viability of calves derived from embryos produced in vitro. Korean Native Cow ovaries were obtained from local slaughterhouse and cumulus-oocyte complexes (COCs) were aspirated from 2 to 8 mm follicles. Selected COCs were matured in TCM-199 supplemented with 10% fetal calf serum (FBS), 1 �ML FSH, 10 �ML LH and 1 �ML Estradiol-17� for 20-22 h. In vitro-matured oocytes were fertilized using frozen-thawed percoll separated semen (Day 0) in fer-TALP medium for 20 h. The presumptive zygotes were cultured in CR1aa medium supplemented with 0.3% BSA (before Day 3) or 10%FBS (After Day 3). All types of cultures were made in an incubator at 38.5�, 5% CO2 in air. Statistical analysis was performed using the Chi-square test. Two blastocysts were transferred to the Holstein recipients (n = 1888). The parturition was occurred in total 755 recipients. There was no difference in the abnormality of calves among treatments. The incidence of disease was significantly higher in single calf than twin calves (18.4 vs. 6.7%), in multiparous than nulliparous group (40.0 vs. 9.9%), in eutocia than dystocia group (20.0 vs. 4.8%), in spring and winter groups than summer and autumn groups (20.3, 22.7 vs. 4.3, 0.0%), and in non-vaccinated than vaccinated group (22.7 vs. 1.6%), respectively (p < 0.05). The rate of mortality was significantly higher when transferred into nulliparous than multiparous (22.3 vs. 0.0%), when were dystocia than eutocia group (71.4 vs. 14.1%), when were non-midwifery than midwifery (45.0 vs. 13.6), when delayed midwifery than earlier midwifery (31.6 vs. 11.5%), and when were non-vaccinated than vaccinated group (28.0 vs. 9.8%), respectively (P < 0.05). The present study suggested that the viability of bovine calves derived from in vitro was affected by the recipient parity, parturition treatment technique and vaccination. This study was supported by the BIO-GREEN 21 PROGRAM.

321 DEVELOPMENT IN VIVO AND IN VITRO OF PORCINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC INJECTION OF A FREEZE - DRIED SPERM HEAD

The present study investigated the development in vivo and in vitro of in vitro matured porcine oocytes injected with a freeze-dried (FD) boar sperm head. In mice, DNA damage was induced during the holding period after rehydration and before sperm injection (Wakayama, T. and Yanagimachi, R. 1998, Nat. Biotechnol., 16, 639–641). Here, we examined the relationship between duration of rehydration of FD sperm and in vitro development of FD sperm-injected porcine oocytes. We also assessed the in vivo developmental competence of the injected oocytes after embryo transfer. Ejaculated boar spermatozoa were suspended in Pig-FM (Suzuki, K. et al. 2002, Int. J. Androl. 25, 84–93) and sonicated for 1 min to separate sperm heads from the tails. An aliquot (100 μL) of the sperm suspension was put into a glass tube and then pre-cooled at −40°C for 6 h. Each tube was attached to a freeze-dry system (DuraDry μP, FTS Systems, Stone Ridge, NY, USA) for 12 h. The ampules were closed and stored at 4°C for more than 7 days before use. For rehydration, 100 μL of distilled water was added into the ampules. In Experiment I, we injected FD sperm heads which were kept for 0–60, 60–120, or 120–180 min after rehydration. At 1 h after the injection, the injected oocytes were stimulated with a DC pulse and cultured for 6 days. The rate of blastocyst formation and the number of cells in the blastocysts were examined. Embryos after in vitro fertilization (IVF) were evaluated as a control. As shown in Table 1, the rates of blastocyst formation were not different (by χ2 test) for duration of rehydration and the control. However, the cell numbers of FD groups were lower (P < 0.05; by Student's t-test) than that in the control. In Experiment II, oocytes injected with a single FD sperm head and stimulated were transferred to both oviducts of a total of ten recipient gilts. Two recipients were diagnosed as pregnant at Day 30 of gestation. At Day 39, one of the pregnant recipients had an abortion, and two fetuses were recovered. The other pregnancy was not maintained. The results suggest that oocytes fertilized with a single FD sperm head have competence to be implanted and to develop to the early fetal stage, and also that the duration for rehydration does not influence in vitro developmental ability in pigs. Table 1. Effects of the duration from rehydration of freeze-dried sperm heads to the injection of the heads into in vitro matured oocytes on in vitro development of the oocytes in pigs

97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

Offspring resulting from transfer of cryopreserved embryos in camel (Camelus dromedarius)

The dromedary embryos, collected at hatched blastocyst stage, survived freezing and thawing in the presence of a high concentration of ethylene glycol (7.0 mol/L) with sucrose (0.5 mol/L) and direct plunging in liquid nitrogen. The rate of survival, as judged by the morphological appearance of the embryos after thawing, was high (92%). The transfer of frozen-thawed embryos into the recipients during the breeding ( n = 20) and non-breeding season ( n = 25) resulted in two and one pregnancy, respectively. One of the two pregnant recipients, with embryos transferred during the breeding season, delivered a normal healthy male calf at term. To our knowledge, this offspring is the first camelid produced following transfer of a frozen-thawed embryo.

Impact of Prior Pregnancy on Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) can cure adults with hematologic malignancies, but results in significant morbidity and mortality. Graft-vs.-host disease (GVHD) is a major complication; attempts to reduce the risk of GVHD include selecting donors based on several characteristics, including parity, a criterion which has been controversial. This retrospective cohort study using data from the Center for International Blood and Marrow Transplant Research (CIBMTR) is the first multi-center analysis of the effects of donor and recipient parity on outcomes of HSCT in the modern transplant era.Methods: We studied patients at least 18 years old who received a non-T-cell-depleted, myeloablative HLA-identical sibling HSCT between 1995 and 1999, for acute lymphoblastic or myelogenous leukemia or chronic myelogenous leukemia. The study endpoints included acute and chronic GVHD, overall survival, and relapse. There were 2,626 patients who met inclusion criteria and had complete information on both donor and recipient pregnancy status. Donors and recipients were categorized as: males, nulliparous females, or parous (one or more pregnancies) females.Analysis: We compared all possible combinations of donor and recipient pregnancy status (9 groups in total); the reference group was male donor/male recipient pairs. Multivariate Cox proportional hazards regression was used to adjust for other prognostic factors. Because multiple groups were compared, significant p-values were considered to be less than or equal to 0.006.Results: After controlling for important patient-, disease-, and transplant-related covariates, the risk of chronic GVHD was significantly increased in parous female donor/male recipient pairs (HR 1.56, 95% CI 1.23 – 1.94, p < 0.0001). Neither donor nor recipient parity had an impact on overall survival, acute GVHD, or relapse risk.Conclusions: This multi-center retrospective registry study showed that parous female donors resulted in a significantly increased risk of chronic GVHD in male recipients, but without concomitant reduction in relapse. Thus, H-Y antigens may be important targets of GVHD, but not of a graft-versus-leukemia effect. As when selecting unrelated donors, avoidance of parous female donors, particularly for male patients, in HLA-identical sibling transplants is recommended when possible.

Risk factors for syngeneic graft-versus-host disease after adult hematopoietic cell transplantation

AbstractSyngeneic graft-versus-host disease (sGVHD) has been described after hematopoietic cell transplantation (HCT) but remains poorly defined. We retrospectively reviewed adult syngeneic HCTs at our center (1980-2002) for sGVHD to investigate incidence, morbidity, and risk factors with a primary focus on parity. Among 119 transplantations, there were 21 cases of biopsy-proven sGVHD. The cumulative incidence was 18%, with multiorgan involvement in 6 cases and 1 death. sGVHD was more frequent when the donor was parous (32%) than nulliparous (9%) or male (13%;P= .03) and when the recipient was parous (31%) than nulliparous (7%) or male (13%;P= .02). Other univariable risk factors included older age (P< .01), busulfan/melphalan/thiotepa conditioning (P< .01), interleukin-2 (P= .02), HLA-A26 (P= .03), and more recent transplantation year (P< .01). Overall, risk factors were similar to those described in GVHD. Although an independent effect of parity could not be completely separated from other factors, donor and recipient pregnancy history merits further investigation.

Management of pregnancy in transplant recipients

Transplantation offers the best prospect of pregnancy in fertile women with various types of end-stage organ disease. Based on data from the USA National Transplantation Pregnancy Registry, >70% of posttransplant pregnancies have a successful live birth. Greatest experience has been seen in transplants involving the kidney, followed by the liver and then heart. Most pregnancies do not affect graft function significantly but are associated with significant obstetric problems such as spontaneous abortions, premature deliveries, low birthweight, intrauterine growth retardation, preeclampsia, etc. Cesarian section is required in 30% to 50% of patients, due to obstetric or maternal concerns. Neonatal death is rare, but 30% to 50% of live births have complications. Thus, management requires a multidisciplinary team. Similar data have been documented in developed Asian countries such as Japan and Singapore. Although fertility is restored early (<6 months), the current recommendation is to wait for ≥1 year, if not 2 years, after transplantation before allowing pregnancy. Patients without evidence of graft dysfunction and hypertension are more likely to have successful outcomes. Acute rejections may occur but the incidence does not appear to be increased dramatically. Treatment should be to similar to that of nonpregnancy treatment. Further pregnancies may be considered in instances of good outcome for the graft, fetus, and mother. Significant experience and safety has been accumulated in treatment using cyclosporine, azathioprine, prednisolone, and tacrolimus. However, few data are available with regard to the newer immunosuppressants, such as mycophenolate mofetil, and thus these agents are not recommended. Most live births have normal growth and development. Although there is much information available on posttransplant pregnancy it is imperative that each center maintains its own registry of pregnancy and offspring outcomes.

Birth of African Wildcat Cloned Kittens Born from Domestic Cats

In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two sources of oocytes were compared, fusion rate was higher using in vivo-matured oocytes as recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitro-matured oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two groups: recipients (n = 24) receiving < or =25 embryos and recipients (n = 26) receiving > or =30 embryos. Twelve recipients (46.2%) receiving > or =30 embryos were diagnosed to be pregnant, while no pregnancies were established in recipients receiving < or =25 NT embryos. Also, to determine the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n = 17), versus day 5 (n = 4) or day 6 (n = 3) was significantly different. Of the 12 pregnant recipients, nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%) from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first wild carnivores to be produced by nuclear transfer.

Birth of African Wildcat Cloned Kittens Born from Domestic Cats

In the present study, we used the African Wildcat (Felis silvestris lybica) as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of African Wildcat (AWC) fibroblast cell nuclei with domestic cat cytoplasts. Cloned embryos were produced by fusion of a single AWC somatic cell to in vivo or in vitro enucleated domestic cat cytoplasts. When the two sources of oocytes were compared, fusion rate was higher using in vivo–matured oocytes as recipient cytoplasts, but cleavage rate was higher after reconstruction of in vitro–matured oocytes. To determine the number of reconstructed embryos required per domestic cat recipient to consistently establish pregnancies, AWC cloned embryos were transferred within two groups: recipients (n = 24) receiving ≤25 embryos and recipients (n = 26) receiving ≥30 embryos. Twelve recipients (46.2%) receiving ≥30 embryos were diagnosed to be pregnant, while no pregnancies were established in recipients receiving ≤25 NT embryos. Also, to determine the influence of length of in vitro culture on pregnancy rate, we compared oviductal transfer on day 1 and uterine transfer on day 5, 6, or 7. Pregnancy rates were similar after transfer of embryos on day 1 (6/12; 50.0%), day 5 (4/9; 44.4%), or day 6 (2/5; 40.0%) to synchronous recipients, but the number of fetuses developing after transfer of embryos on day 1 (n = 17), versus day 5 (n = 4) or day 6 (n = 3) was significantly different. Of the 12 pregnant recipients, nine (75%) developed to term and fetal resorption or abortion occurred in the other three (25%) from day 30 to 48 of gestation. Of a total of 17 cloned kittens born, seven were stillborn, eight died within hours of delivery or up to 6 weeks of age, and two are alive and healthy. Perinatal mortality was due to lung immaturity at premature delivery, placental separation and bacterial septicemia. Subsequent DNA analysis of 12 cat-specific microsatellite loci confirmed that all 17 kittens were clones of the AWC donor male. These AWC kittens represent the first wild carnivores to be produced by nuclear transfer.

Implantation and pregnancy following in vitro fertilization and the effect of recombinant human relaxin administration in Macaca fascicularis.

Implantation and early pregnancy, and the potential effects of the reproductive-hormone relaxin, were examined in the cynomolgus macaque (Macaca fascicularis) following in vitro fertilization and embryo transfer. Mature oocytes were collected from regularly cycling, female cynomolgus monkeys subjected to ovarian superovulation using recombinant human FSH and hCG. Oocytes fertilized in vitro were cultured to the 4- to 8-cell stage, slow-cooled, and stored in liquid nitrogen before thawing and embryo transfer. Regularly cycling recipients were administered recombinant human relaxin or vehicle for 21 days through the peri-implantation period (Day 0 = pump implantation), during which time the thawed embryos were transferred (Day 7). Endometrial thickness and the number of gestational sacs were monitored by ultrasound at three time points (Days 7, 21, and 28). The number of days of placental sign (implantation bleeding) in pregnant females and menses in nonpregnant females were also recorded. Implantation (gestational sacs/embryo transferred) and multiple pregnancy (multiple gestations/ pregnant recipient) rates were slightly higher in relaxin-treated recipients compared to vehicle-treated recipients. Administration of relaxin was associated with increased implantation bleeding in pregnant females. Endometrial thickness was increased in relaxin-treated recipients at Days 7 and 28 compared to Day 0, but these differences were not observed at the same time points in vehicle-treated females. Systemic administration of recombinant human relaxin in an in vitro fertilization/embryo transfer setting was associated with effects consistent with a role for this hormone in endometrial physiology in primates.

Pregnancy in heart transplant recipients

The aim of this report is to present data from Italian cardiac transplant centers assessing pregnancy after cardiac transplantation. Our retrospective survey included 10 pregnancies occurring in 7 patients during January 1991 to February 2002. Eight pregnancies were completed successfully and 2 abortions were reported (frequency rate 20%). No complications were observed during pregnancy or after delivery. Of 8 infants studied, 6 (75%) were born at term and 2 (25%) pre-term. One baby presented congenital talipes valgus. Pediatric development was uneventful. The data from the literature and our series show that a multidisciplinary approach is mandatory. The course of pregnancy is usually normal and the maternal and fetal outcomes are usually favorable. Although no fetal malformations have been reported, prolonged follow-up of these infants is required.

Fetal death: comparative aspects in large domestic animals

Although the majority of pregnancy failures occur during the embryonic period, reports indicate that approximately 5% of detected pregnancies are lost during the fetal period, underlining the fact that fetal death is a substantial cause of economic loss. However, examination for fetal development or death during pregnancy is not performed routinely in domestic animals, and reference curves for normal fetal growth are, therefore, scarce. In this paper, the numerous possible causes of fetal death are reviewed briefly, with emphasis on the role of placental problems in fetal death and impaired fetal viability. In this respect, the role of placental insufficiency as a cause of pregnancy loss in twin pregnancies in monotocous species is well known, whereas the abnormal placental development leading to retarded fetal growth during pregnancies in recipients of in vitro produced (IVP) or nuclear transfer (NT) embryos has been less extensively documented. Fetal viability or death can be evaluated using hormonal, chemical and ultrasonographic parameters. For example, the viability of the feto-placental unit can be examined by measuring maternal plasma concentrations of oestrone sulphate or the placental proteins, including pregnancy-associated glycoprotein (PAG) and pregnancy-specific protein B-60 (PSPB-60). Low concentrations of any of these three indicate either no pregnancy, or if pregnancy was confirmed earlier, fetal death and abnormally high or low levels can indicate fetal abnormality. Ultrasound can be used to examine the fetal heart rate (FHR), the incidence of fetal movements (FM), the appearance of fetal fluids and the development of the fetus and placenta. However, although abnormal FHRs have been correlated to subsequent fetal death, it is important to remember that there is a large physiological variation in FHR at the end of gestation, due to different behavioural states and differences in FM patterns. Although monitoring fetal viability and death using hormonal and ultrasonographic evaluations is possible during pregnancy in domestic animals, there is considerable physiological variations in the ‘normal’ values. Therefore, suitable combinations of tests need to be identified and more accurate reference values generated before such approaches can be considered reliable for monitoring the status of individual fetuses.

Renal function and lipid metabolism in pregnant renal transplant recipients

Objective: To estimate renal function and lipid metabolism in pregnant renal transplant recipients. Study design: The study covered 64 women during the third trimester of pregnancy including 33 renal transplant recipients (the study group) and 31 healthy women (the control group). Serum concentrations of uric acid, urea, creatinine, electrolytes, total protein, albumin, acid-base balance and blood cell count were examined to assess renal function. Moreover, the levels of the following lipid metabolism parameters were estimated: (1) total lipids (TL), (2) total LDL fraction (TLDL), (3) total cholesterol (TCh), (4) free cholesterol (fCh), (5) free/total cholesterol (fCh/TCh) ratio, (6) phospholipids (PhL), (7) total cholesterol/phospholipids (fCh/PhL) ratio, (8) triglycerides (TG), (9) HDL-cholesterol (HDL-Ch), (10) LDL-cholesterol (LDL-Ch) and (11) LDL-Ch/HDL-Ch ratio. ‘The effect of immunosuppressants (cyclosporine, prednisone and azathioprine) on serum lipid levels was estimated in the study group. The mean maternal age, gestational age and BMI did not differ in both groups. Results: Pregnant renal transplant recipients presented mild renal insufficiency during the third trimester resulting in the increase in serum level of uric acid (P<0.001), urea (P<0.001), creatinine (P<0.001), and Cl− (P<0.001). Proteinuria (1.19±1.9 g/24 h) leading to hypoproteinemia (P<0.001) and hypoalbuminemia (P<0.05) confirmed renal function impairment in the study group. Additionally, the diagnosis of renal insufficiency was supported by mild acidosis reflected by a drop in pH (P<0.001). standard HCO3− (P<0.001) and base excess (P<0.001). The women with renal grafts presented vital lipid metabolism disturbances illustrated by the elevated levels of: (1) TL by 72% (P<0.001), (2) TLDL by 21% (P<0.001), (3) TCh by 16% (P<0.001), (4) fCh by 34% (P<0.001), (5) fCh/TCh ratio by 21% (P<0.001), (6) PhL by 28% (P<0.001), (7) TG by 53% (P<0.001), (8) LDL-Ch by13% (P<0.05) and (9) LDL-Ch/HDL-Ch ratio by 23% (P<0.001). No difference in HDL-Ch level between the two groups was found. Hyperlipidemia in pregnant kidney recipients was associated with immunosuppressive treatment and depended on cyclosporine treatment regimen. Treatment with azathioprine and prednisone was associated with elevated serum levels of examined lipids. Conclusion: Serum lipid abnormalities are significantly influenced by the administered dosages of immunosuppressants.

Comparison of two vitrification methods for cryopreservation of porcine embryos

The aim of this study was to compare two vitrification methods of porcine perihatching blastocysts with regard to the success of transfer of these embryos to the recipients. Expanded, hatching, or hatched blastocysts were recovered post mortem from superovulated donors in 5.5 to 6.0 days after artificial insemination of donor gilts with homospermic doses. In protocol VS I, the embryos in perihatching developmental stage were equilibrated in a culture medium H-MEMD with 10% v/v of glycerol (1.37M solution of glycerol in medium) for 10 min and placed in a vitrification medium for 1.5 min max. (vitrification medium contained 50% v/v 2M sucrose in tridistilled water, 30% v/v of glycerol, and 20% v/v of foetal calf serum &amp;ndash; FCS). Then they were dropped with micropipette and stored in liquid nitrogen vapour. For protocol VS II, we used H-MEMD culture medium supplemented with 20% v/v of FCS, 25% v/v ethylene glycol, and 25% v/v dimethyl sulphoxide (DMSO). Embryos were equilibrated for 10&amp;nbsp;min in a mixture of the vitrification medium and culture medium (1 : 1), and were kept in the vitrification medium for 1.5&amp;nbsp;minutes. Then they were dropped with micropipette and stored in liquid nitrogen vapour. Embryos were thawed by immersing the drop with the embryo in H-MEMD culture medium with 0.8M sucrose for 10 minutes. After thawing and washing in the medium with sucrose, all embryos were washed three times in a fresh medium and prepared for transfer. Recipients were synchronized either using Regumate-feeding followed by treatment with PMSG and HCG (gilts) or using piglet weaning (sows &amp;ndash; 1st and 2nd parity). Recipients showing standing heat at the time of donor insemination were used for laparoscopic and non-surgical ET on day 5.5&amp;ndash;6.0 of the cycle. The fraction of viable embryo vitrified under VS I or VS II protocol was 85% and 80%, compared to 95% in control fresh embryos (P &amp;gt; 0.05). Pregnancy of recipients was 57.3% (5/7), 67.0% (4/6) for VS I or VS II group and 42.7% (10/23) for control (P &amp;lt; 0.001). We can conclude on the basis of our data that both protocols for vitrification yielded similar results and can be used for cryopreservation of porcine embryos. &amp;nbsp;

Pregnancy outcome after renal transplantation

Aims The aim of our study was to evaluate the frequency and the outcome of pregnancies in renal transplant recipients at our center. Methods This study involved the retrospective analysis of 405 childbearing female renal recipients for presence of risk factors, the outcome of pregnancy, and maternal and fetal complications. Results Fourty-four pregnancies occurred in 41 patients (10.8%). Mean age at transplantation was 23.6 ± 6.3 years (range, 12–38 years). Only in 5 pregnancies were there no risk factors. In 13 (29.5%) pregnancies, the previous creatinine level was >1.5 mg/dL, in 16 (36.45%), proteinuria was >500 mg/24 hours; 29 (65.9%) were hypertensive; 14 (31.8%) had a time between transplantation and pregnancy less than 2 years (mean time, 35.5 ± 30.9 months; range, 3–120 months). The outcomes were 27 (61.4%; 11 term and 16 premature delivery) successful pregnancies, 6 (13.6%) spontaneous abortions, 10 (22.7%) therapeutic abortions, and 1 (3.2%) fetal death. Pre-eclampsia occurred in 9 (20.4%) pregnancies and eclampsia in 1 (2.2%). The mean weight of the offspring was 2195 ± 490 g (range, 1300– 2980 g). There were 2 cases of acute fetal distress and 1 oligodramnios. Median creatinine level was 1.0 (range, 0.4–3.0) mg/dL before conception and 1.2 (range, 0.7–9.0) mg/dL 6 month after pregnancy ( P < .001). The long-term patient and graft survival rates were similar for pregnant versus nonpregnant recipients in the childbearing age. Conclusion Most pregnancies were successful, although the premature delivery rate was high (36.4%). Only 5 conceptions occurred in the absence of risk factors. Pregnancy did not impair the patient and graft survival during long-term follow-up.