Therapeutic effect of human umbilical cord mesenchymal stem cells on tubal factor infertility using a chronic salpingitis murine model.

BACKGROUND: The study was conducted to evaluate the application of human umbilical cord mesenchymal stem cells (hUCMSCs) in the treatment of tubal factor infertility (TFI) caused by Chlamydia trachomatis, and investigate their effect on fertility in animal models of chronic salpingitis. METHODS: In this study, we investigated the therapy effects of the transplantation of hUCMSCs in tubal factor infertility using a chronic salpingitis murine model which induced Chlamydia trachomatis. Twenty rats were divided into two groups: control group (n = 10) and treatment group (n = 10). hUCMSCs were given to mice after exposure to C. trachomatis for 4 weeks. After treatment for 4 weeks, five mice were randomly selected from each of the two groups to sacrifice and we examined the organ morphology and pathology, inflammatory cytokines, proliferation, and apoptosis in fallopian tube (FT).The remaining five mice from each group were caged 2:1 with male mice for another 4 weeks, the numbers of pregnant mice and the mean number of pups in the different groups were enumerated and calculated. RESULTS: Intravaginal inoculation of hUCMSCs alleviated hydrosalpinx of the oviduct. EdU-labeled hUCMSCs are located at the interstitial site of the fallopian tube. Macrophage (F4/80) infiltration was significantly reduced in the treatment group compared with the control group and expression levels of the anti-inflammatory cytokine IL10 were increased after hUCMSCs treatment. Furthermore, mRNA and protein expression levels of PCNA and Caspase-3 were increased and decreased, respectively, in the hUCMSCs' treatment group compared with the control. Moreover, hUCMSCs' transplantation improved murine fertility. CONCLUSIONS: Anti-inflammatory and anti-apoptotic effects of hUCMSCs may play an important role in TFI. Our data suggest that hUCMSCs' transplantation contributed to the repair of tubal injury and improvement of fertility, providing a basis for assessing the contribution of stem cells in the oviduct for direct repair of the tube to assist reproduction.

Effect of tubal explants and their secretions on bovine spermatozoa: modulation of ROS production and DNA damage.

Although low levels of reactive oxygen species (ROS) play a physiological role in maintaining sperm function, an increase in ROS generation above these levels may result in the induction of sperm membrane and DNA damage. The main objective of this study was to determine whether bovine oviducal explants (TU) and their conditioned media (CM) have a modulatory effect on the production of ROS, and consequently, on sperm DNA integrity. Thawed sperm were exposed to bovine TU and to CM obtained from the ampullar and isthmal regions after 4 and 12h, and DNA damage and intracellular ROS production was assessed by TUNEL and DHE and SYTOX Green, respectively. Co-incubation of spermatozoa with oviducal explants from the ampullar region (TUa) for 4h resulted in a statistically significant increase in the percentage of spermatozoa with DNA damage compared with controls (P=0.0106), and this increase was positively correlated with ROS levels. Conversely, although the incubation of spermatozoa with explants and conditioned media from the isthmal region (TUi and CMi, respectively) for 12h resulted in an increase of spermatozoa with DNA damage compared with controls (P<0.0001), this increase was not correlated with ROS levels. In conclusion, significant oxidative stress may take place in the oviduct, particularly during short-term incubation, and this may be related to changes in the antioxidant factors present in the oviducal cells and secretions. A redox imbalance in pro-oxidants and antioxidants in the oviduct may lead to oxidative stress and sperm DNA damage.

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.

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.

Restoration of endocrine function and fertility with orthotopic tubal-ovarian allotransplant as the anatomical-functional unit in rabbits.

The aim of this study was to re-establish endocrine and reproductive function in tubal-ovariectomized rabbits using orthotopic tubal-ovary allotransplants (OT-OA). Fifty-five New Zealand White nonconsanguineous rabbits were used and allocated into five experimental groups: Each group comprised donors submitted to right salpingo-oophorectomy that served as the donated allograft. In group 1 no cyclosporin (CsA) was administered and rabbits were submitted to left salpingo-oophorectomy (LSO). Group 2 was the allotransplant group and did not receive CsA. Group 3 was the allotransplant group with CsA. They were submitted to bilateral salpingo-oophorectomy followed by OT-OA. CsA of 10 mg/kg/day was administered to rabbits for the first 21 days and 5 mg/kg/day for the remainder of the study. Group 4 received CsA just as for group 3 and was submitted to LSO. During the study the following were measured: estradiol (E2), progesterone (P4), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and histopathological study of the uteri, tubes, and ovaries. Groups 1, 3, and 4 showed a significant increase in serum E2 and P4 levels corresponding to the second week of gestation, compared to group 2 (p < .05). Group 2 showed a significant increase in serum LH and FSH levels during week 10 compared to serum LH and FSH levels in groups 1, 3, and 4 (p < .05). In group 3, three rabbits did not reject the allotransplant, one rabbit became pregnant, and endocrine function was re-established in two rabbits. It can be concluded that OT-OA together with CsA administration re-establishes endocrine function and fertility.

Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation.

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.

Restoration of fertility in oophorectomized rats after tubo-ovarian transplantation.

Improved microsurgical techniques for en bloc vascularized adnexal isograft in the rat is described. The graft of the right ovary together with its fallopian tube and upper third of uterus was transplanted orthotopically with end-to-side anastomoses between the donor aortic segment and recipient aorta and between the donor vena cava segment and recipient inferior vena cava, with end-to-end anastomosis of the donor and recipient uterus in a syngeneic, bilaterally oophorectomized rat. All transplantations were successful in terms of immediate vascular patency rate (10/10, 100%). Evidence of resumed ovarian function was obtained in 9 out of 10 rats (9/10, 90.0%) by histological demonstration of the vaginal smear, in which pregnancies were achieved in six rats (6/10, 60.0%) and six litters of healthy offspring were delivered 9 weeks later after transplantation. These results suggest that microsurgical ovarian transplantation provide a new and potential experimental model for the study of fertility restoration in humans.

Fertility after intact ovary transplantation.

Transplantation surgery, which is limited by the supply and short-term viability of fresh donor organs, would be revolutionized if these could survive freezing, but early claims of cryopreservation were never realized. Here we describe the successful transplantation in rats of ovaries, fallopian tubes and the upper segment of the uterus en bloc after storage in liquid nitrogen.

Stable engraftment of human female genital mucous membrane xenografts on SCID mice.

We developed a model in which full-thickness human genital mucous membranes (fallopian tubes, endometrium) were heterotopically xenografted into the skin of severe combined immunodeficient (SCID) mice. The transplanted tissue retained its human phenotype for at least 4 weeks including the glandular epithelium, the lamina propria, and main parts of the grafted vessels. By using an occlusive chamber filled with covering phosphate-buffered saline we created a system that protected the moist human epithelial surface. This system will allow the study of the interaction of test substances, or of invasive, pathogenic microorganisms, with epithelial cells and other cellular components of the human genital mucosa under in vivo conditions.

Juxtaposition of contralateral ovary and fallopian tube to allow pregnancy in unicornuate uterine anomaly.

Infertile patients who have undergone unilateral oophorectomies or salpingectomies or those with uterine anomalies may require juxtaposition of a remaining fallopian tube and contralateral ovary. A 28-year-old woman with previous left oophorectomy for endometriosis was found to have a left unicornuate uterus during subsequent infertility evaluation. Laparotomy and resection of a right rudimentary horn and juxtaposition of the left fallopian tube and the right ovary was performed. A large endometrioma was also resected from the right ovary. The patient conceived during the second month postoperatively. A simple procedure for juxtaposing a contralateral fallopian tube and ovary is described. Careful evaluation of the pelvic organs should be performed before removal of an ovary in a young woman.

[Transposition of the fallopian tube as a therapeutic possibility in special cases of tubal sterility]. / Die Transposition der Tube als Therapiemöglichkeit in besonderen Fällen von tubarer Sterilität.

This is a report on a new method of microsurgical transposition of the Fallopian tube. It was developed to deal with congenital malformations or with the different anatomic remnants after pelvic surgery or pelvic inflammatory diseases. The tube is mobilised by severing the mesosalpinx with the unipolar microelectrode without damaging the longitudinal vessels. After mobilisation, the bends of the tube disappear and the length increases almost doublefold. Then the tube can be laid to the contralateral ovary without any tension. The tube must be fixed to the ovary in such a way, that the fimbrial end is mobile enough to lay onto the ovary during ovulation. Up to now, six patients with longstanding tubal sterility were operated upon. Three of them became pregnant, one twice. There were no ectopic pregnancies and no abortions.

Demonstration of delayed hypersensitivity in Chlamydia trachomatis salpingitis in monkeys: a pathogenic mechanism of tubal damage.

The role of delayed hypersensitivity in the pathogenesis of Chlamydia t trachomatis salpingitis was studied in the monkey "pocket" model. Pigtailed monkeys (Macaca nemestrina) were sensitized by inoculation of live C. trachomatis organisms (E/UW-5/Cx) into subcutaneous pockets containing salpingeal autotransplants. At 21 days, affinity-purified recombinant C. trachomatis heat-shock protein (rhsp60) was injected into pockets either previously sensitized with C. trachomatis or not sensitized in the same monkey. Delayed-type hypersensitivity reaction was observed, characterized by mononuclear cell infiltration with peak reaction at 48 h. Injection of rhsp60 into the pockets of a naive animal did not induce inflammation. This study showed that C. trachomatis infection in monkeys induced delayed hypersensitivity, which is mediated by hsp60. Histologic findings of the salpinx were consistent with delayed hypersensitivity reaction observed in ocular C. trachomatis infection, further suggesting a similar pathogenesis for both salpingitis and trachoma.

Ovarian function, tubal viability and pregnancy after tubo-ovarian transplantation in the rabbit.

A rabbit model was developed for microsurgical en-bloc vascularized tubo-ovarian allograft. Transplantation of tube and ovary from donors to tubo-oophorectomized recipients was technically successful in 50 rabbits. After surgery, animals were randomly allocated into two groups: animals in group A received cyclosporine for immunosuppression; animals in group B did not receive immunosuppressive therapy. In group A, 16 animals survived the transplant procedure and six animals became pregnant (38%). No pregnancies were obtained among animals in group B. In group A, plasma concentrations of ovarian and pituitary hormones were similar to those found in non-transplanted animals. Our results show firstly that tubo-ovarian transplantation is technically feasible, and secondly that cyclosporine improves not only tubal viability but also ovarian function after transplantation.

[Tubo-ovarian transplantation in the treatment of sterility. Experimental research]. / Il trapianto tubo-ovarico nel trattamento della sterilita'. Ricerca sperimentale.

Microsurgical transposition of fallopian tube and ovary has the potential of being an efficient therapeutic treatment in patients with tubal sterility. The Authors present their experience of microsurgical adnexal transplantation in rabbit by two different techniques: the first procedure by microvascular anastomosis of the ovarian vessels, the second one without vascular pedicle. Function is evaluated at various time after grafting by: exploratory laparotomy on day 30 to establish whether circulation to the grafts was still maintained; macroscopic and microscopic examination of ovaries and fallopian tubes. The microvascular techniques prove highly reliable in terms of immediate vascular patency rate but it is disappointing that 50% of the autografts has failed with blocked vessels by day 30. Perhaps this is due to the difficult techniques in anastomosing the ovarian vessels of small caliber. In spite of these outcomes the vascularized autografts were viable and functional after transplantation in contrast with the non-vascularized tubo-ovarian grafts which all failed. This experience encourages to believe that the microsurgical technique could be employed for homograft transplantation in woman with extensive ovarian and tubal damages.

Free autologous graft of fimbria to ampulla with subsequent oviductal function in the rabbit.

To test the hypothesis that a free, nonvascularized graft of fimbria would retain structure and function, fimbriae were transplanted autologously in 17 rabbits. Subsequently, the transplanted fimbrial tissue survived, with patency in almost all the animals, was capable of transporting surrogate cumulus oophorus but had reduced ovum capture capability. That the transplanted rabbit fimbria retains a degree of structure and function suggests that the technique warrants an investigation in women.

Chlamydia trachomatis oculogenital infection in the subcutaneous autotransplant model of conjunctiva, salpinx and endometrium.

The subcutaneous pocket model of salpingeal, endometrial, and conjunctival autografts for studying Chlamydia trachomatis infection in monkeys is described. Portions of the salpinx that were transplanted included fimbria, ampulla, and isthmus. The model is an extension of the original model which consists of either salpingeal fimbria or conjunctive autografts. Transplantation of the ampulla portion of the Fallopian tube enabled us to increase the number of pockets or test sites. Salpingeal and conjunctival autografts could be established during a single surgery. In addition, it is possible to autotransplant endometrium and provoke endometritis. The autografts were shown to be susceptible to C. trachomatis infection. Preliminary rechallenge experiments showed infection of the subcutaneous transplants may induce immunity, indicating the model may be used for immunity and vaccine studies. Simultaneous transplantation of different parts of the oviduct, endometrium, and conjunctive should expand the usefulness of the subcutaneous model in other studies on mixed infections or immune responses to infection.