Preclinical report on allogeneic uterus transplantation in non-human primates.

STUDY QUESTION: Is it possible to perform allogeneic uterus transplantation (UTx) with a donation from a live donor in a non-human primate species and what immunosuppression is needed to prevent rejection? SUMMARY ANSWER: Allogeneic UTx in the baboon is a donor- and recipient-safe surgical procedure; immunosuppression with induction therapy and a triple protocol should be used. WHAT IS KNOWN ALREADY: UTx may become a treatment for absolute uterine factor infertility. Autologous UTx models have been developed in non-human primates with reports on long-term survival of the uterine grafts. STUDY DESIGN, SIZEAND DURATION: This experimental study included 18 female baboons as uterus donors and 18 female baboons as uterus recipients. The follow-up time was 5-8 weeks. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Uterus retrieval was performed with extended hysterectomy including bilateral uterine and internal iliac arteries and ovarian veins. After UTx, with vascular anastomoses unilateral to the internal iliac artery and the external iliac vein, the uterus recipients received one of the following: no immunosuppression (n = 4); monotherapy (oral slow release tacrolimus) (n = 4) or induction therapy (antithymocyte globulin) followed by triple therapy (tacrolimus, mycophenolate, corticosteroids; n = 10). Surgical parameters, survival, immunosuppression and rejection patterns were evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: The durations of uterus retrieval and recipient surgery were around 3 and 3.5 h, respectively. The total ischemic time was around 3 h. All the recipients and the donors survived the surgery. All the recipients presented rejection to some extent within the first weeks following UTx. In one recipient, the uterus was of normal appearance at the end of the study period. In spite of occasional high (>60 ng/ml) blood levels of tacrolimus, there was no evidence of nephrotoxicity. LIMITATIONS AND REASONS FOR CAUTION: This initial non-human primate allogeneic UTx study indicates that further research is needed to optimize immunosuppression protocols in order to avoid uterine rejection. WIDER IMPLICATIONS OF THE FINDINGS: The findings suggest that allogeneic UTx in primate species is feasible but continued work on this issue is needed. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by the Swedish Research Council, ALF University of Gothenburg, Hjalmar Svensson Foundation and by Jane and Dan Olsson Research Foundation. The authors do not have any competing interest.

Uterus transplantation in a non-human primate: long-term follow-up after autologous transplantation.

BACKGROUND: Uterus transplantation (UTx) may provide the first available treatment for women affected by uterine infertility. The present study aimed to further develop a surgical technique for autologous UTx in a non-human primate species and to assess long-term function. METHODS: Female baboons (n= 16) underwent autologous transplantation of the uterus with the Fallopian tubes and ovaries, performed with a previously published surgical technique (n= 6, Group 1) or using a modified technique (n= 10; Group 2). The uterine arteries were dissected to the proximal end of the anterior branch (Group 1) or the entire (Group 2) internal iliac artery, and the ovarian veins were dissected to the crossing over the ureter (Group 1) or further cranially to include greater lengths and patches of the cava/renal vein (Group 2). Back-table preparation created common venous and arterial ends with arterial anastomosis either end-to-side to the left external iliac artery (Group 1) or end-to-end to the left internal iliac artery (Group 2). RESULTS: Overall short-time survival of the animals was 88% (66% in Group 1 and 100% in Group 2). Of all the operated animals, 75% (66% in Group 1 and 80% in Group 2) resumed ovarian cyclicity. Regular menstruation after UTx was demonstrated only in Group 2 (60%). Menstruating animals (n= 6) were each exposed to timed mating for ≥5 menstrual cycles, but pregnancy did not occur. Adhesions and tubal blockage were seen in post-mortem analysis. CONCLUSIONS: The modified UTx model of Group 2 is a safe procedure and shows resumed long-term uterine function in a majority of the animals, although pregnancy could not be demonstrated.

Uterine transplantation research: laboratory protocols for clinical application.

The aim of this review is to summarize the state-of the-art methods that are used in clinical organ transplantation today, as well as the major findings of recent experimental uterus transplantation (UTx) research regarding organ donation/retrieval, ischemic preservation, surgical techniques for anastomosis, immunosuppression and pregnancy. Absolute uterine factor infertility lacks treatment despite the major developments in infertility treatment and assisted reproduction. Concerning uterine factor infertile patients, genetic motherhood is only possible through gestational surrogacy. The latter can pose medical, ethical and legal concerns such as lack of control of life habits during surrogate pregnancy, economic motives for women to become surrogate mothers, medical/psychological pregnancy-related risks of the surrogate mother and uncertainties regarding the mother definition. Thus, surrogacy is non-approved in large parts of the world. Recent advances in the field of solid organ transplantation and experimental UTx provide a favourable and safe background in a scenario in which a human clinical UTx trial can take place. Protocols based on animal research over the last decade are described with a view to providing a scientifically guided approach to human UTx as an experimental procedure in the future.

Fertility after autologous ovine uterine-tubal-ovarian transplantation by vascular anastomosis to the external iliac vessels.

BACKGROUND: Transplantation of the uterus has been suggested as a treatment of uterine factor infertility. This study investigates whether the sheep uterus can resume its capacity to harbour normal pregnancies after autotransplantation by vascular anastomosis. METHODS: From 14 ewes, the uterus, excluding one uterine horn, was isolated along with its oviduct and ovary and preserved ex vivo and then transplanted back with end-to-side anastomosis of the vessels of the graft to the external iliac vessels. After recovery, the ewes underwent surgical examination and serum progesterone measurements to ascertain healing and ovarian activity. Afterwards, five autotransplanted and five control ewes were placed with a ram for mating. Caesarean sections were performed before the estimated term of pregnancy and data on fetal measures were compared. RESULTS: Of the 14 ewes, seven survived surgery with ovarian activity intact and grafts showing normal appearance. Mating occurred in four of five transplanted ewes and in five out of five controls, and three transplanted animals and five control animals conceived. In one transplanted ewe, torsion of the uterus was observed after spontaneous initiation of labour. Foeti from transplanted mothers were comparable in size to those of controls. CONCLUSIONS: Despite the encountered complications, this is the first report to demonstrate fertility and pregnancies going to term after autotransplantation of the uterus in an animal of a comparable size to the human.

Uterus transplantation in the baboon: methodology and long-term function after auto-transplantation.

BACKGROUND: Techniques for uterus transplantation (UTx) have been developed in rodent/domestic animals towards future clinical introduction of UTx to treat uterine factor infertility. The aim of this study was to extend the UTx research into a non-human primate species by developing surgical techniques for uterus retrieval and transplantation in the baboon. METHODS: Female baboons (n = 15) underwent surgery, with the initial five animals used for studies of pelvic vascular anatomy. Retrieval surgery included isolation of the ovarian veins and the uterine arteries together with the anterior branches of the internal iliacs. The utero-tubal-ovarian specimen was removed, flushed and kept ex vivo for 2 h when the two arterial ends and two venous ends were anastomosed side-to-side to construct one arterial and one venous end. These were, at auto-transplantation, anastomosed end-to-side to the external iliacs and the animals (n = 10) were evaluated concerning cyclicity and later by laparoscopy/laparotomy. RESULTS: The total duration of organ retrieval, backtable preparation and transplantation was around 6 h with an overall ischaemic time of the specimen of about 3 h. One animal died due to cardiomyopathy. Five out of the nine surviving animals resumed cyclicity, as a sign of re-established ovarian function. Only two out of these five animals exhibited resumed menstruation, indicating re-established ovarian and uterine function. Laparoscopy confirmed normal-sized uteri in these two animals. CONCLUSIONS: This study demonstrates the feasibility of UTx by vascular anastomosis in a non-human primate species. The low success rate demonstrates the complexity involved in UTx surgery and the need for further methodological developments.

Plasma concentrations of nitrate during the menstrual cycle, ovarian stimulation and ovarian hyperstimulation syndrome.

BACKGROUND: Nitric oxide (NO) is predominantly a locally acting mediator, affecting several functions in the human female reproductive tract. In vivo, it is quickly metabolized to its stable end product nitrate, which is cleared by the kidney. METHODS AND RESULTS: The aim of the present study was to evaluate possible fluctuations of plasma nitrate concentrations during the menstrual cycle, ovarian stimulation as well as ovarian hyperstimulation syndrome (OHSS). During the menstrual cycle (n = 19 women) the mean nitrate concentrations were between 26.7 and 29.5 micromol/l at all stages except for the day of ovulation, when the concentrations were significantly (P < 0.001) increased (mean 37.2 micromol/l +/- 2.0). Significantly lower concentrations of plasma nitrate (P < 0.01) were measured at the end of gonadotrophin-releasing hormone (GnRH) down-regulation (24.6 micromol/l +/- 1.4) compared with the concentrations found at day 8 of follicle-stimulating hormone (FSH) stimulation (34.9 micromol/l +/- 2.6) and at the day of human chorionic gonadotrophin (HCG) (35.6 micromol/l +/- 3.3). The concentrations of nitrate (33.4 micromol/l +/- 3.4) in women with OHSS (n = 13) were similar to those seen 5 days after embryo transfer (33.2 micromol/l +/- 2.3). CONCLUSIONS: The results indicate that NO synthesis is increased at the time of spontaneous ovulation. GnRH treatment inhibits NO synthesis, while NO production is not increased in women with OHSS.

Plasma levels of free vascular endothelial growth factor(165) (VEGF(165)) are not elevated during gonadotropin stimulation in in vitro fertilization (IVF) patients developing ovarian hyperstimulation syndrome (OHSS): results of a prospective cohort study with matched controls.

OBJECTIVE: To evaluate whether differences in plasma vascular endothelial growth factor(165) (VEGF(165)) concentrations exist during gonadotropin stimulation in IVF patients developing severe ovarian hyperstimulation syndrome (OHSS) compared to matched controls. STUDY DESIGN: Prospective cohort study with comparison of 15 OHSS cases with 30 matched (age, follicle numbers, pregnancy) controls. Unpaired students t-test was used to evaluate differences between the OHSS and control group and correlations were calculated with Pearson's test. RESULT(S): Plasma levels of VEGF(165), at four time-points from start of gonadotropin stimulation to embryo transfer (ET), were compared and related to steroid levels and ultrasound data. There were no differences between OHSS and control patients in plasma VEGF(165) levels at any of the four time points, which were compared. The mean levels were between 53--83 pg ml(-1) and 64--83 pg ml(-1) in the OHSS and control group, respectively. Positive correlations existed between total number of follicles, number of large (>15 mm) follicles and VEGF(165) at day of oocyte aspiration and between VEGF(165) and progesterone at ET in the control group, but not in the OHSS group. CONCLUSION(S): Patients developing OHSS do not have raised plasma VEGF(165) levels during gonadotropin stimulation. The lack of positive correlation between VEGF(165) levels and follicle numbers/progesterone in the OHSS group, suggests a disruption in OHSS of the normal controlled follicular VEGF expression.

Low peripheral blood levels of the immunosuppressive cytokine interleukin 10 (IL-10) at the start of gonadotrophin stimulation indicates increased risk for development of ovarian hyperstimulation syndrome (OHSS).

Our hypothesis was that patients developing ovarian hyperstimulation syndrome (OHSS) might have a disturbed responsiveness or delayed activation of the immunosuppresive cytokine system. In a prospective cohort study, women (n=428) undergoing in vitro fertilisation (IVF) treatment were subjected to repeated blood sampling and collection of clinical data. Fifteen patients, who developed severe OHSS, were compared with matched (age, follicle numbers, pregnancy) control patients. Samples of serum and plasma were collected throughout the stimulation and up to 7 days after embryo transfer as well as during hospitalisation for OHSS. Levels of IL-4, IL-10, IL-13, oestradiol and progesterone were measured. Significantly lower levels of IL-10 were seen at the start of gonadotrophin stimulation in OHSS patients, with an increase seen after the development of OHSS. In these OHSS patients, a negative correlation between IL-10 levels and number of follicles at time of aspiration existed, but there were no correlations between steroid and IL-10 levels. Levels of IL-13 and IL-4 were low in both groups and did not change during stimulation. The lower levels of IL-10 at start of stimulation in OHSS patients, as compared with controls, may be of pathophysiological importance by allowing for an enhanced Th-1 type immune response during gonadotrophin stimulation and thereby increased and generalised inflammation. The increase in IL-10 after development of OHSS indicates that IL-10 at that time is induced in a systemic attempt to suppress the inflammation of OHSS.

Peripheral blood concentrations of inhibin B are elevated during gonadotrophin stimulation in patients who later develop ovarian OHSS and inhibin A concentrations are elevated after OHSS onset.

Ovarian hyperstimulation syndrome (OHSS) is a serious side-effect of controlled ovarian stimulation. Inhibin A and inhibin B, as putative predictors of OHSS development in the same stimulation cycle, were evaluated. A cohort of 428 in-vitro fertilization (IVF) patients was followed. Fifteen patients with severe OHSS were compared with matched (age, follicle number) controls. Serum samples were obtained at five time points from the start of ovarian stimulation until >/= 3 days post-embryo transfer and analysed with specific enzyme-linked immunosorbent assays. Inhibin A in the OHSS group showed a continuous increase with a significant elevation 3 days prior to oocyte aspiration (ASP-3) and onwards. Maximal concentrations were detected at embryo transfer and the concentrations remained high at >/= 3 days post-embryo transfer. Inhibin A concentrations in the control group showed a transient elevation (significant increase at ASP and embryo transfer). Inhibin A in the OHSS group was significantly higher than in controls only at the time point where OHSS had developed (>/= 3 days post-embryo transfer), and declined during OHSS treatment. Overall, there was a positive correlation between the number of follicles and inhibin A concentrations at ASP-3 until embryo transfer in the control group but not in the OHSS group. The concentrations of inhibin B in both groups increased from the start of ovarian stimulation, with peak values at ASP-3, and then a decline. Inhibin B was significantly higher in OHSS patients at ASP-3 and at ASP. Inhibin B at ASP-3 was correlated with the total number of follicles in both the OHSS group and the control group.

Prospective study of the clinical and laboratory parameters of patients in whom ovarian hyperstimulation syndrome developed during controlled ovarian hyperstimulation for in vitro fertilization.

OBJECTIVE: To compare patient characteristics and clinical and laboratory parameters in patients in whom ovarian hyperstimulation syndrome (OHSS) develops with those in whom it does not develop. DESIGN: Prospective cohort study. SETTING: Reproductive medicine unit at a university medical center. PATIENT(S): All patients undergoing IVF (n = 428) who received controlled ovarian hyperstimulation during a 6-month period. INTERVENTION(S): Prospective data collection. MAIN OUTCOME MEASURE(S): Patient characteristics (age, body mass index, medical history, smoking habits) and clinical and laboratory data obtained during controlled ovarian hyperstimulation were evaluated in patients who had severe OHSS, any degree of OHSS, or a significant risk of OHSS and compared with the remaining populations. RESULT(S): Severe OHSS developed in 18 patients (4.2%) and mild or moderate OHSS developed in 7.3%. As a group, all the patients with OHSS were significantly younger, received lower doses of gonadotropins, had ovaries containing a higher number of total and large follicles, had a higher number of retrieved oocytes, and had a higher pregnancy rate than the patients without OHSS. The patients with severe OHSS also had an increased prevalence of allergy (56% versus 21%) and were more likely to ultimately give birth. CONCLUSION(S): The observed differences may be useful in elucidating the pathophysiology of OHSS and identifying patients who are at increased risk for OHSS.

Complement anaphylatoxin C3a and C5a formation in premature children with respiratory distress.

UNLABELLED: Premature children (n = 25) with respiratory distress (RD) were studied regarding complement activation and formation of the anaphylatoxins C3a and C5a. Blood samples were drawn on admission to the paediatric intensive care unit. In 18 of the patients RD was accompanied by other perinatal complications like pneumothorax or intracerebral haemorrhages. Seven of the premature children had RD without such complications. Preterm children with RD and with peri- and postnatal complications such as pneumothorax or intracerebral haemorrhage had increased concentrations in plasma of the anaphylatoxins C3a and C5a compared with preterm children with RD without these complications. There was a positive correlation between the plasma C3a and C5a concentrations in the preterm children. CONCLUSION: The present study indicates that isolated RD will appear without signs of complement activation and that complications like pneumothorax or intracerebral haemorrhages are associated with release of the anaphylatoxins C3a and C5a.

Immunoglobulin subclasses and prophylactic use of immunoglobulin in immunoglobulin G subclass deficiency.

Persistent low serum levels of one or several immunoglobulin G (IgG) subclasses can be found in a high proportion of adult patients with increased susceptibility to infections. It is hard to envision that the low subclass level in itself is responsible for this susceptibility because healthy blood donors have been described who are completely devoid of certain subclasses in serum. This apparent discrepancy may be partly explained by the observation that most subclass-deficient patients have underlying aberrations in T-cell and B-cell interaction and function that may impair their capacity to compensate for even minor deficiencies. A prospective blind crossover study of the effect of prophylactic Ig substitution therapy was done in 43 adult patients with IgG subclass deficiency. The patients were randomized to receive 1 year of therapy with intramuscular Ig 25 mg/kg/wk or 1 year of saline injections. A significant protective effect of the prophylactic Ig therapy was seen with a great reduction in the number of days of infection. In addition, several immunologic parameters were altered after 1 year of Ig therapy. Nineteen of the patients later were included in an open study using 50 mg/kg/wk of Ig. After 6 months of treatment, significant protection against infection was observed, with a reduction of 6.2 days in the number of days per month with infection. This marked effect of prophylactic Ig suggests that the Ig aberrations seen in IgG subclass-deficient patients contributed to their susceptibility to infection. The effect of 25 mg/kg/wk was much less pronounced than that of 50 mg/kg/wk, and normal serum IgG subclass levels were not achieved even during the higher-dose therapy. However, it seems likely that subcutaneous or intravenous administration of larger doses of Ig would allow for more efficient therapy.

Opinions on treatment of women with habitual abortion based on investigations for blocking antibody and autoantibodies.

Three hundred and thirty-seven women with habitual abortion of unknown etiology were studied for cellular reactivity and blocking antibody in one-way mixed lymphocyte culture. Their sera were investigated for anti-cardiolipin antibodies, antinuclear antibodies, and antibodies against DNA, and the activated partial thromboplastin time (APTT) and complement levels of their plasma were determined. Increased anti-cardiolipin antibody levels were demonstrated in 77 (22%) of the 337 women, all of whom were considered healthy and had no signs of autoimmune disease. Most patients with high anti-cardiolipin antibody levels displayed lowered values of complement factor C4. According to our experiences, the mere occurrence of anti-cardiolipin antibody in women with habitual abortion is no absolute cause for treatment with prednisolone, not even in cases with greatly elevated anti-cardiolipin values. Therapy with prednisolone and acethylsalicylic acid (ASA) during pregnancy should be given to those women who have high levels of anti-cardiolipin antibodies concomitant with high APTT values, low values of complement C4, and strong blocking antibody. Anti-cardiolipin antibody has been investigated during pregnancy in 136 normal pregnant women, 11 of whom (8%) were positive at any sampling occasion, but only one of whom (1%) had high levels. Evidently the development of anti-cardiolipin antibody is no normal feature of pregnancy among Swedish women and thus the high frequency found among healthy Swedish women with habitual abortion remains unexplained. We have introduced an immunization program of leukocyte transfusions in habitual abortion. The development of previously absent blocking antibody seems to be a valuable prognostic sign of possible success for immunization therapy against habitual abortion.

Immunoglobulin subclass (IgG3) restriction of anti-P and anti-Pk antibodies in patients of the rare p blood group.

Serum antibodies against globoside and ceramide trihexoside, two major glycolipids in human erythrocytes, were investigated in 29 individuals with the rare p blood group. Antibodies of the IgM and of the IgG3 classes were detected. In AB Rh(-) blood donors, who were not of the p blood group, low levels of antibodies of the IgM class were found against P and Pk, whereas no antibodies were detected in cord blood. The increased number of spontaneous abortions and stillbirths observed among the p individuals may relate to the presence of IgG3 antibodies, because such antibodies pass the placental barrier and are efficient in complement activation and in mediating antibody-dependent cytotoxicity.