Functional Ovarian Tissue Research Articles

Identifying ovarian tissue in the bitch using anti-Müllerian hormone (AMH) or luteinizing hormone (LH)

Reliable methods for determining whether or not a bitch has ovarian tissue present are needed for cases with unknown neutering status. Vaginal cytology consistent with heat is indicative of functional ovarian tissue. Other methods are required when the bitch is not presented in suspected heat. Progesterone can be analyzed during 2 months after suspected heat. During other stages, assays for the analyses of anti-Müllerian hormone (AMH) and luteinizing hormone (LH) have been used. The AMH assay is expected to give detectable concentrations (positive) in bitches with ovarian tissue, and the LH assay should give negative results in intact bitches, except during the pre-ovulatory LH peak. The aim of the present study was to study the diagnostic efficiency for detecting ovarian tissue in bitches using an AMH assay developed for human samples, and a semi-quantitative rapid immune migration (RIM™) LH assay developed for use in dogs. An AMH concentration of ≥0.1 μg/L, and an LH concentration of ≤1 μg/L, was set as the cut-off for presence of ovarian tissue. Client or staff owned bitches were included (N = 125). There were 73 intact bitches that were classified as being in heat (N = 25); in luteal phase (N = 12); or in anestrus (N = 36), and 52 spayed bitches that showed no clinical signs of estrogen influence. In total 64 of the 73 intact bitches (88%) were correctly identified using AMH, and 70/73 (96%) intact bitches were correctly identified using the LH assay. Excluding bitches in heat, the corresponding figures were 42/48 (88%) for AMH and 48/48 (100%) for LH. Of the 52 spayed bitches, 51 (98%) were correctly identified using the AMH assay and 49 (94%) were correctly identified using the LH assay. In this population, the predictive value of a positive AMH for intact bitches was 98%, and of a negative AMH for spayed bitches was 85%. Excluding bitches in heat, the predictive value of a negative LH test for intact bitches was 94%, and the predictive value of a positive LH test for identifying spayed bitches was 100%. It was concluded that analyses of AMH and LH are useful for detecting ovarian tissue in bitches, but that low concentrations of AMH may be obtained in intact bitches, classifying them as spayed. For LH, bitches in suspected estrus should not be tested to avoid the pre-ovulatory LH-surge, that otherwise may cause intact bitches being incorrectly identified as spayed.

Subcutaneous ovarian tissue transplantation in nonhuman primates: duration of endocrine function and normalcy of subsequent offspring as demonstrated by reproductive competence, oocyte production, and telomere length.

The main purposes of the study were to investigate the endocrine function of ovarian tissue transplanted to heterotopic subcutaneous sites and the reproductive competence and telomere length of a nonhuman primate originating from transplanted tissue. Ovarian cortex pieces were transplanted into the original rhesus macaques in the arm subcutaneously, in the abdomen next to muscles, or in the kidney. Serum estradiol (E2) and progesterone (P4) concentrations were measured weekly for up to 8years following tissue transplantation. A monkey derived from an oocyte in transplanted ovarian tissue entered time-mated breeding and underwent controlled ovarian stimulation. Pregnancy and offspring were evaluated. Telomere lengths and oocytes obtained following controlled ovarian stimulation were assessed. Monkeys with transplants in the arm and abdomen had cyclic E2 of 100pg/ml, while an animal with arm transplants had E2 of 50pg/ml. One monkey with transplants in the abdomen and kidney had ovulatory cycles for 3years. A monkey derived from an oocyte in transplanted tissue conceived and had a normal gestation until intrapartum fetal demise. She conceived again and delivered a healthy offspring at term. Controlled ovarian stimulations of this monkey yielded mature oocytes comparable to controls. Her telomere length was long relative to controls. Heterotopic ovarian tissue transplants yielded long-term endocrine function in macaques. A monkey derived from an oocyte in transplanted tissue was reproductively competent. Her telomere length did not show epigenetically induced premature cellular aging. Ovarian tissue transplantation to heterotopic sites for fertility preservation should move forward cautiously, yet optimistically.

Restoration of fresh cat ovarian tissue function by autografting to subcutaneous tissue: A pilot study

Ovarian tissue transplantation could be a valuable technique for the preservation of endangered animals. The domestic cat affords an adequate experimental model for studies aimed at wild felids due to its phylogenetic similarity. Thus, this pilot study evaluated the efficacy of cat ovarian tissue autotransplantation to a peripheral site. Three adult queens were submitted to ovariohysterectomy. The ovaries were fragmented into eight pieces; two were fixed as a control and six were transplanted to subcutaneous tissue of the dorsal neck. Grafts were monitored weekly by ultrasound and fecal samples collected daily in order to monitor estradiol levels. Grafts were recovered on Days: 7, 14, 28, 49 and 63 post-transplantation for histological analyses. One graft was maintained in one animal for 8 months. A total of 2466 ovarian follicles were analyzed: 1406 primordial and 1060 growing follicles. All animals presented antral follicles in one or more of the grafts. The percentage of morphologically normal primordial follicles was always higher than 80%, except for Day 7 transplants. Although the proportion of growing follicles increased after transplantation, there was a general decrease in the percentage of morphologically normal growing follicles from Day 7 onwards. All animals demonstrated at least three estradiol peaks during the 63-day period, and one animal exhibited estrous behaviour on three occasions. Hormonal peaks directly correlated with the visualization of antral follicles (by ultrasound and/or histology) and the observation of estrous behaviour. Long-term results on one female showed the concentration of 37.8 pg/mL of serum estradiol on Day 233 post-grafting and the female exhibited estrous behaviour on several occasions. This graft showed one antral follicle, one luteinized follicle and two preantral follicles. In conclusion, cat ovary autotransplantation to the subcutaneous tissue restored ovarian function, with hormone production and antral follicle development, over both short and long term periods. This could be a valuable technique in the evaluation of ovarian cryopreservation methods in felids. Once the technique is shown successful, it may be applied in allografts or xenografts between different feline species.

Hyaluronic acid-based hydrogel scaffold without angiogenic growth factors enhances ovarian tissue function after autotransplantation in rats

One of the problems encountered during ovarian transplantation is that the number of primordial follicles in the grafts is considerably reduced 2 d after transplantation due to post-transplantation ischemia. This study investigates if the use of hyaluronic acid-based hydrogel (HABH) with and without vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) could prevent or minimize ischemia-induced follicle loss during ovarian autotransplantation and thereby restore ovarian tissue function in the rat model. In this study, twenty four female rats were subjected to bilateral ovariectomy and were randomly divided into 3 groups for ovarian tissue autotransplantation. Group A included rats with ovarian tissue without HABH, VEGF and bFGF, group B comprised rats with ovarian tissue encapsulated with HABH and group C had rats with ovarian tissue encapsulated with HABH containing VEGF and bFGF. Three days after transplantation, the grafts were assessed through histological and hormonal analyses. Apoptotic, angiogenic and maturation genes expressions were also analyzed. The mean number of follicles in all developmental stages increased in group B (P < 0.05). The level of FSH decreased in group B (P < 0.05) whereas, the expression level of VEGF gene increased in group B (P < 0.05). No significant changes were observed in the expression levels of maturation and apoptotic genes in all groups. In conclusion, ovarian encapsulation with HABH alone can prevent or minimize ischemia-induced follicle loss, preserve the follicular pool, promote follicular survival, facilitate angiogenesis, and restore hormone levels. However, its efficiency in a clinical setting and in comparison with other hydrogels needs further investigation.

Restoration of ovarian tissue function and estrous cycle in rat after autotransplantation using hyaluronic acid hydrogel scaffold containing VEGF and bFGF

This study investigates the effect of hyaluronic acid (HA) containing VEGF and bFGF on restoration of ovarian function after ovarian autotransplantation. Twenty-four rats were randomly divided into three groups for ovarian autotransplantation: group A (ovaries without HA, VEGF and bFGF), group B (ovaries encapsulated with HA) and group C (ovaries encapsulated with HA containing VEGF and bFGF). The grafts were assessed using vaginal smears, histological, hormonal, and the genes expression analysis. The duration of first estrous cycle was shorter in group C than in group A (p < 0.01). The mean number of primordial follicles was protected in group C. The level of estradiol was higher in group A than in group C (p < 0.01). The expression level of Cellular-Myelocytomatosis (C-Myc) in group C was lower than in group B (p < 0.05). HA containing VEGF and bFGF can ensure follicular survival, decrease apoptosis and recover ovarian function after auto-transplantation.

Dysgerminoma, Pyometra of the Stump and Mammary Carcinoma in a Female Dog with Ovarian Remnant Syndrome-Case Study

Ovarian remnant syndrome (ORS) represents the presence of functional ovarian tissue after routine castration of the bitch, caused either by surgeon error or the presence of anomalous ovarian tissue. The piece of ovary that remains after the surgery, may continue to develop and become functional, secreting hormones as estrogen and progesterone which cause signs of pro-estrus or estrus, even pseudocyesis. All those signs cause consternation among owners, who thought that the reproductive activity in their dog will be completely abolished. One mixed-breed, 6 years old bitch, 27.3 kg, spayed 8 months before, was presented with purulent vaginal discharge and mammary lumps, after the animal has been treated by her veterinarian with Proligestone to suppress the persisting estrus signs. Our investigations included vaginal cytology, vaginoscopy, the examination of the mammary chain, ultrasound, radiography, serum chemistry, blood count and histopathological examinations. The ultrasound revealed a filled uterine stump and the presence of a mass caudal the right kidney, which was suspected as an ovarian neoplasm. The examination of the mammary chain revealed masses in right M 3, M4, M5 glands and in the left M2 gland. The bitch underwent surgery, and the results of the histopathological examination were: dysgerminoma, pyometra, and the mammary masses were classified as carcinomas. In this paper we wanted to underline the fact that the Ovarian Remnant Syndrome may imply serious clinical consequences which can even lead to the death of the animal. We recommend as a method of treatment for this condition the surgical removal of the remnant tissue.

Impact of the cryopreservation technique and vascular bed on ovarian tissue transplantation in cynomolgus monkeys.

The aim of this study was to determine the best combination in terms of cryopreservation techniques and vascular bed preparation before grafting in order to obtain functional ovarian tissue after transplantation. Five cynomolgus monkeys were used. Strips from 10 ovaries were cryopreserved, 5 by vitrification (V), and 5 by slow-freezing (SF). Pieces of fresh ovarian tissue were used for controls. After 1 month, the strips were autografted to two different vascular beds, healed (HB) or freshly decorticated (FDB), constituting four study groups: SF-HB, SF-FDB, V-HB, and V-FDB. These were compared to fresh tissue. After 6 months, the ovaries were removed and several parameters analyzed: follicle quality, stage, density, proliferation, apoptosis, functionality, vascularization, and fibrosis. Mixed effect linear regression models were built to assess the impact of cryopreservation and vascular bed preparation on ovarian tissue viability and functionality. p values were adjusted for multiple testing using the Benjamini-Hochberg method, and q values < 0.20 were considered significant in order to achieve a 20% false discovery rate. Compared to fresh tissue, no difference was observed in the percentage of morphologically normal follicles, while a significant increase was noted in the follicle proliferation rate (41%, q = 0.19), percentage of antral follicles (12%, q = 0.14), and number of vessels per area (3.3 times, q = 0.07) in the V-FDB group. Vitrification associated with FDB vascular bed preparation is the best combination to obtain functional autografted ovarian tissue. Further studies are nevertheless required, with confirmed pregnancies and live births before introducing the procedure into clinical practice.

Динаміка структурних змін оваріальної тканини при ішемії

In this work it was investigated the dependence of the correlation of folliculogenesis and endocrine function of ovarian tissue on the degree of structural damage and oocyte volume changes during ischemia. It was shown that after 2 hours of ischemia at 37 °C the morphological transformation of the structural components of the ovarian tissue were reversible. In case of restoration of blood flow conditions by heterotopic transplantation estradiol level of animals was 25,9 ± 5,18 pg/ml, progesterone--18,48 ± 3,69 ng/ml, significantly higher than the castrated animals-recipients. Supplement of the incubation medium by the fetal bovine serum lead to decreasing by 5-7% in the volume of oocytes of growing follicles during in ischemia and reduced steroidogenic function of ovarian tissue after perfusion. Increased time of the ischemia up to 4 hours was founded in irreversible morphological transformation, reduce the volume of oocytes by 40% and the formation of sclerosed tissue after transplantation of the ischemic fragments of ovarian tissue.

Robotic Removal of an Ovarian Remnant for Chronic Pelvic Pain

Ovarian remnant syndrome is a rare condition in which functional ovarian tissue presents as a pelvic mass and/or pelvic pain after prior intended removal of one of both ovaries with or without hysterectomy. An ovarian remnant presents as not only a diagnostic challenge, but a surgical one as well. We present a case of a patient with chronic left lower quadrant pain after a C-hysterectomy and left salpingo-oophorectomy who was found to have a 3 x 2 x 4 cm residual ovarian tissue anterior to the psoas muscle.

VEGF and bFGF increase survival of xenografted human ovarian tissue in an experimental rabbit model

The aim of this study is to determine whether vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) could increase the survival of xenografted human ovarian tissue in an experimental rabbit model. Fresh human ovarian tissue was xenotransplanted into the back muscle of 25 castrated female New Zealand rabbits for 6weeks with the immunosuppression of FTY720 (2mg/kg/d). Rabbits were randomly divided into five experimental groups: (A) graft and host treatment with VEGF (50ng/ml); (B) graft and host treatment with bFGF (100ng/ml); (C) graft and host treatment with VEGF(50ng/ml) + bFGF (100ng/ml); (D) graft and host treatment with normal saline; (E) control group, no treatment. 4weeks after transplantation, human menopausal gonadotropin (HMG) 10IU was administered every second day in group A, group B, group C and group D for 2weeks. Graft survival was assessed by graft recovery rate, histological analysis, immunohistochemical staining for CD31 and Ki-67expression, TUNEL assay. After 6weeks of grafting, the number of CD31-positive stained cells increased significantly in group A, group B and group C compared to the control group. All groups showed strong Ki-67 immunostaining in ovarian stroma. Only one rabbit in group C retained the grafts' follicles. Grafting resulted in relative lower fibrosis in group A and group C compared to the control group. Apoptosis was significantly lower in group C compared to the control group. Fresh human ovarian cortex grafted into the back muscle of rabbit can sustain part of ovarian tissue function with the immunosuppression of FTY720, although follicle number diminishes significantly after grafting. The administration of VEGF and bFGF, especially the combination of them, may trigger angiogenesis, reduce apoptosis and fibrosis, increase survival in transplanted human ovarian tissue.

Viability of human ovarian tissue confirmed 5 years after freezing with spontaneous ice-formation by autografting and chorio-allantoic membrane culture

To achieve optimal and uniform outcomes, slow cooling protocols for human ovarian tissues generally initiate ice formation at high sub-zero temperatures (−6 to −9°C). The aim of the study was to investigate the function of ovarian tissue that had unintentionally self seeded at −20°C during the freezing step, by examining its development following chicken embryonic chorioallantoic membrane (CAM) grafting and after transplantation back to the patient. Ovarian tissue was frozen in 6% (v/v) dimethyl sulfoxide, 6% (v/v) ethylene glycol and 0.15M sucrose which had self-seeded at −20°C. Five years after cryopreservation, 8 pieces were thawed and transplanted back to the patient. Two small (1×2×1mm) pieces of this thawed tissue were cultured in a CAM-system for 5days to assess the tissue viability. The autografted ovarian tissue re-established spontaneous menstrual bleeding within five months and raised serum 17-β Estradiol from 19 to 330pg/ml. Ultrasound revealed a dominant follicle at the site of the transplanted tissue in the follicular phase after the menstrual bleed. Analysis of the CAM cultured tissue established that 88% of the primordial follicles are degenerated and there was limited in growth of blood vessels. In conclusion, in spite of the damage caused by the cryopreservation with spontaneous ice-formation the viability could be confirmed by CAM culture and the restoration of ovarian function after auto-transplantation.

In vitro generation of three-dimensional functional ovarian structures

INTRODUCTION: The ovary contains cells that secrete hormones, which maintain female sexual characteristics as well as egg production. Loss of ovarian tissue function due to various endocrine and fertility disorders requires chronic estrogen and progesterone administration. While conventional hormone replacement therapy is able to maintain female sexual characteristics, this therapy is unable to restore ovarian tissue function, which includes the production of eggs. This is critical for premenopausal women who desire to conceive.

Function of ovarian tissue after long-term storage

Ovarian tissue cryopreservation is one of the options available to preserve fertility in cancer patients and to allow them to conceive when they have overcome their primary disease. The publication of a report by Anderson and colleagues documenting live births in three women arising from ovarian cortical strips that had been transplanted more than 7years previously represents an apparent further advance in this field.

Cyst aspiration with gonadotropin suppression in ovarian remnant syndrome with ureteral obstruction: A case report

Background: Ovarian remnant syndrome (ORS) is characterized by functional ovarian tissue following bilateral salpingo-oophorectomy. ORS presents with pelvic pain or mass, but may also present with urinary tract symptoms resulting from enlarging residual tissue. Case: A 37 year old woman presented with pelvic pain radiating to her flank due to ureteral obstruction from an enlarging ovarian cyst in ORS. Transvaginal ultrasound guided, ovarian cyst aspiration accompanied by leuprolide acetate gonadotropin suppression resulted in acute and chronic relief of ureteral obstruction. Conclusion: This case demonstrated successful treatment of ureteral obstruction caused by an ovarian cyst in a patient with ORS via cyst aspiration and leuprolide acetate gonadotropin suppression. This treatment is a viable alternative for management of ORS, but sacrifices pathologic diagnosis when compared to traditional surgical resection.

Effects of gonadotropin-releasing hormone administration on the pituitary-gonadal axis in male and female dogs before and after gonadectomy

GnRH-stimulation tests were performed in 14 female and 14 male client-owned dogs of several breeds, before and 4 to 5 mo after gonadectomy. The aim of the study was to obtain more insight into the pituitary-gonadal axis in intact and neutered dogs and to establish reference values. Basal plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) concentrations were increased significantly after gonadectomy in both bitches and male dogs. In both males and females ranges of the basal plasma FSH concentrations, before and after gonadectomy, did not overlap as opposed to the overlap in ranges of the basal plasma LH concentrations. Before gonadectomy basal plasma LH concentrations were lower and basal plasma FSH concentrations were higher in bitches than in male dogs. After gonadectomy these basal values did not differ significantly. GnRH administration before gonadectomy resulted in an increase in plasma LH and FSH concentrations in both genders. GnRH administration after gonadectomy produced an increase only in plasma LH concentrations in both genders, and a just significant increase in plasma FSH in castrated male dogs. GnRH administration before gonadectomy resulted in a significant increase in plasma testosterone concentration in both genders. In males ranges of basal and GnRH-stimulated plasma testosterone concentrations before and after gonadectomy did not overlap. Basal plasma estradiol concentrations were significantly higher in intact males than in castrated males and their ranges did not overlap. The basal estradiol concentrations in bitches before and after ovariectomy were not significantly different. At 120 min after GnRH administration, ranges of plasma estradiol concentration of intact and ovariectomized bitches no longer overlapped. In conclusion, basal plasma FSH concentration appears to be more reliable than basal plasma LH concentration for verification of neuter status in both male and female dogs. The basal plasma testosterone concentration appears to be reliable for verification of neuter status in male dogs. The plasma estradiol concentration at 120 min after GnRH administration can be used to discriminate between bitches with and without functional ovarian tissue.

Long-term followup of autologous, non-human primate ovarian tissue translants and reproductive competence in offspring born from ovarian tissue transplantation

OBJECTIVE: Ovarian tissue freezing & transplantation is a novel way to preserve fertility. Human pregnancies have resulted, but there are no data if these offspring can conceive, and little is known on how long ovarian grafts function. Here we 1) compare the endocrine function of ovarian tissue transplanted to various sites & 2) see whether a non-human primate, born from an egg derived from transplanted ovarian tissue, is fertile.

Production of Donor-Derived Offspring from Cryopreserved Ovarian Tissue in Japanese Quail (Coturnix japonica)1

Cryopreservation of avian ova and embryos is challenging because of the yolky structure of the egg. As an alternative, with the development of effective cryopreservation protocols, ovarian tissue cryopreservation could be used for cryobanking for birds. Pieces of ovarian tissue of week-old Japanese quail (Coturnix japonica) were frozen at 0.5 degrees C/min in a programmable freezer or vitrified by immersion in liquid nitrogen. Straws containing slow-frozen samples were thawed in ice water, and vitrified samples were removed from the vials and transferred into sucrose, with the concentration lowered in sequence at room temperature. Cell viability of tissue was estimated by trypan blue assay, and tissue histology was examined by light microscopy. Frozen-thawed or vitrified-warmed tissue from WB (recessive plumage color) chicks was transplanted into week-old ovariectomized QO (wild-type plumage) chicks, with some chicks receiving fresh tissue as a control group. At sexual maturity, QO recipients were mated to WB males, and the production of WB offspring demonstrated successful cryopreservation and transplantation. Donor-derived offspring were obtained from the ovarian tissue that had been cryopreserved by either slow-freezing or vitrification. The vitrification protocol used in this study showed better outcomes at each level of evaluation. This study demonstrated that the function of ovarian tissue in avian species can be successfully preserved at subzero temperatures and recovered by transplantation. The vitrification protocol is recommended because of high efficiency and overall simplicity.

Sonographic changes after laparoscopic cystectomy compared with three-stage management in patients with ovarian endometriomas: a prospective randomized study

Laparoscopic surgery is the gold standard treatment for ovarian endometriomas. The aim of this study was to evaluate the impact of two different laparoscopic methods on sonographic indicators of ovarian reserve in the treated ovary. In this prospective randomized clinical trial, 20 patients with endometriomas were randomly assigned to undergo either laparoscopic cystectomy (Group 1) or the 'three-stage procedure' (Group 2). All patients underwent ultrasound examination preoperatively and 6 months after laparoscopy. Another ultrasound examination was performed 12 months after intervention in order to detect any recurrence. We investigated the alterations in the residual ovarian volume, ovarian vascular supply and antral follicle count (AFC) on the ovary with the endometriotic cyst by transvaginal color Doppler ultrasonography. The residual ovarian volume and the lowest pulsatility and resistance indexes were found to be similar between the two groups before and 6 months after laparoscopic intervention. The AFC of the operated ovary was increased significantly (P = 0.002) in Group 2 compared with Group 1 after 6 months. One year after laparoscopy, two recurrences of endometriomas were detected in Group 2, although none were detected in Group 1 (P = 0.47). Ovarian volumes and vascularization were comparable among the two laparoscopic methods. On the contrary, functional ovarian tissue as determined by AFC was higher after the 'three-stage' procedure.

Videolaparoscopia no diagnóstico e tratamento da síndrome do ovário remanescente em uma gata

A síndrome do ovário remanescente é definida como a persistência da atividade ovariana em fêmeas castradas. É decorrente da presença de tecido ovariano acessório no ligamento largo uterino ou por erro na técnica cirúrgica de ovariosalpingohisterectomia ou ovariectomia. O presente trabalho descreve o diagnóstico e tratamento videolaparoscópico de um caso de síndrome de ovário remanescente em uma gata. No acesso, foram utilizados três portais de 5mm nas paredes abdominais direita, esquerda e na linha média ventral. Constatou-se a presença de massa com aspecto de tecido ovariano junto à fossa paralombar esquerda e aumento de volume na fossa paralombar direita, removidos com tesoura de Metzenbaum e cauterização monopolar. O exame histológico da massa extirpada do lado esquerdo confirmou a presença de tecido ovariano. Não se observaram complicações perioperatórias, e a paciente evoluiu sem sinais de recidiva de estro pelo período de pelo menos 24 meses. Conclui-se que a síndrome do ovário remanescente em gatas pode ser diagnosticada e tratada com sucesso por cirurgia laparoscópica.

Ectopic ovary autotransplanted over rectosigmoid colon: a case report

To report a case of an alive ectopic ovary over rectosigmoid colon serosa that was removed by laparoscopy. Case report. Department of Obstetrics and Gynecology. A 30-year-old nulliparous woman. Ultrasonography, laparoscopy, and pathologic assessment. Alive and functional ectopic ovarian tissue in the cul-de-sac over rectosigmoid colon serosa. At pathologic assessment, an alive ectopic ovary with focal infarct areas was determined. The treatment should be excision of the mass. However, in asymptomatic and nulliparous women who have future fertility desire, protecting the ectopic ovary to not decrease the ovarian reserve might be a beneficial option.