The use of near-infrared imaging with indocyanine green in the ovarian tissue transplantation: a case report.

The request for fertility preservation has consistently increased in recent years. To our knowledge this case report is the first to describe the application of near-infrared intraoperative imaging using indocyanine green (NIR-ICG) during ovarian tissue transplantation (OTT), to assist surgeon choosing the site of implantation of ovarian fragments. OTT was performed in a 42-year-old woman using NIR-ICG to evaluate the vascularisation of peritoneal area as the site of implantation for the ovarian graft. we believe this new approach could be useful in identifying the best reimplantation site.

Progesterone-responsive vaginal leiomyoma and hyperprogesteronemia due to ovarian luteoma in an older bitch.

BACKGROUND: This is the first report about a vaginal leiomyoma concomitant with an ovarian luteoma in a bitch. CASE PRESENTATION: A 11-year-old intact female Labrador retriever was referred because of anuria, constipation and protrusion of a vaginal mass through the vulvar commissure. The bitch had high serum progesterone concentration (4.94 ng/ml). Because of the possibility of progesterone responsiveness causing further increase of the vaginal mass and since the bitch was a poor surgical candidate a 10 mg/kg aglepristone treatment was started SC on referral day 1. A computerized tomography showed a 12.7 × 6.5 × 8.3 cm mass causing urethral and rectal compression, ureteral dilation and hydronephrosis. A vaginal leiomyoma was diagnosed on histology. As serum progesterone concentration kept increasing despite aglepristone treatment, a 0.02 ng/mL twice daily IM alfaprostol treatment was started on day 18. As neither treatment showed remission of clinical signs or luteolysis, ovariohysterectomy was performed on referral day 35. Multiple corpora lutea were found on both ovaries. On histology a luteoma was diagnosed on the left ovary. P4 levels were undetectable 7 days after surgery. Recovery was uneventful and 12 weeks after surgery tomography showed a reduction of 86.7% of the vaginal mass. The bitch has been in good health and able to urinate without any complication ever since. CONCLUSIONS: This case demonstrates the importance of identifying progesterone related conditions as well as the importance of judiciously using a combined medical and surgical approach.

Surgery and out-patient data collection and reporting using Filemaker Pro.

OBJECTIVE: The application of an electronic database in clinical practice is used widespread in every field of medicine. The aim of the present study is to illustrate our experience to use a database software for documentation of two of our clinical activities, outpatient hysteroscopy and inpatient gynaecological surgery. PATIENTS AND METHODS: In 2004, we designed two databases, the first one to document surgical procedures in the operating theatre, the second to document outpatient hysteroscopy procedures using FileMaker v.8.5. The data entry interface contains free text fields for patient demographic data and the description of the surgical procedure, supplemented by drop-down lists for items such as clinical findings, procedures, instrumentation, technique, and complications. Copies were filed in the main hospital notes, sent to General Practitioners, and also given to our patients. RESULTS: Since August 2004, we have used our two databases to document 2766 gynaecological operations and 3777 outpatient hysteroscopies. All users particularly liked the dropdown lists as their use greatly reduced the time taken to enter each patient's data. The databases were regularly used to select patients for audit projects and research data collection for prospective studies. CONCLUSIONS: FileMaker is an user-friendly and easily configured software, extremely valuable in everyday clinical work.

Biochemical composition of fetal fluids in at term, normal developed, healthy, viable dogs and preliminary data from pathologic littermates.

A proper canine neonatal assistance, required to reduce the high perinatal loss rate, imply a full knowledge about the fetal-to-neonatal physiology. Because fetal fluids play an important role throughout mammals pregnancy, influencing fetal growth and development, fetal well being, and contributing to guarantee the most suitable environment for the fetus, the knowledge about fetal fluids biochemical composition is of major importance. At first, the biochemical composition of fetal fluids collected by normal developed, healthy and viable newborns, is necessary to depict the normal features, and represent the first step for the further detection of abnormalities associated to fetal/neonatal distress and useful for the early identification of newborns needing special attention, immediately after birth. The present study was aimed to define the biochemical composition of amniotic and allantoic fluids collected from fetus delivered by caesarean section at term of pregnancy. To reduce the possible confounding effect of maternal labor or troubles at parturition, fetal fluids were collected only from puppies born by elective caesaeran section, at term of normal pregnancies. Fetal fluids from 76 puppies, 70 normal and six pathologic newborns, born by elective caesarean section were collected and analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, lactate dehydrogenase (LDH), creatine-kinase (CK), alkaline phosphatase (ALP), creatinine, urea, amylase, lipase, gamma-glutamyl transferase (γ-GT), triglycerides, cholesterol, total proteins, albumin, globulins, glucose, magnesium, potassium, chloride, sodium, calcium, phosphorus and osmolarity. No significant differences were found between biochemical composition of amniotic or allantoic fluid in normal and pathologic newborns, maybe due to the small number of the pathologic puppies. Although some correlations between the two fluids were found (albumin, phosphorus, glucose and triglycerides), the results showed significant differences between the amniotic and allantoic biochemical composition (for all the parameters, except of alanine aminotransferase, triglycerides, cholesterol, albumin, amylase and glucose), suggesting that diverse sources could concur to the final composition of each fluid. A wide variability within and among litters was found for both amniotic and allantoic biochemical composition, and for some parameters an influence of breed body size (amniotic amylase, cholesterol, and allantoic calcium and glucose), maternal parity (amniotic and allantoic CK, glucose, LDH, chloride) and newborn gender (allantoic phosphorus) was found. Further investigations are needed for addressing the origin of each fetal fluid biochemical composition in the dog and also to indeep possible differences in fetal fluids biochemical composition between normal and pathologic puppies, providing potential markers for the quick identification of newborns that need special surveillance and cares immediately after birth.

The efficacy of modified luteal phase support with intramuscular progesterone in IVF/ICSI cycles: a retrospective observational study.

OBJECTIVE: The use of gonadotropin-releasing hormone agonist for ovulation triggering has become an intriguing topic in the last few years. As long as adequate luteal phase support is provided, it may be a valuable alternative to standard hCG triggering, associated with a significant reduction in OHSS incidence. Several luteal phase support options have been proposed, but few studies have addressed the issue of the appropriate route for progesterone administration to women triggered with GnRHa. The aim of the study was to evaluate the effect of GnRHa triggering on IVF/ICSI outcomes, using modified luteal phase support with intramuscular progesterone. PATIENTS AND METHODS: A retrospective study was carried out between January 2014 and December 2015, comparing the reproductive outcome in GnRHa triggered women given modified luteal phase support with intramuscular progesterone (Group A) with the outcome in women triggered with standard hCG (Group B) in IVF/ICSI cycles. RESULTS: 200 (Group A n = 100; Group B n = 100) consecutive normoresponder women were included. No differences with respect to Age, BMI, basal FSH, basal Estradiol and infertility diagnosis were observed between groups. Increased numbers of retrieved oocytes (8.1 ± 3.3 versus 6.8 ± 3.5, p = 0.009) and mature oocytes (5.8 ± 2.6 versus 5.1 ± 2.7, p = 0.03) were detected in Group A compared with Group B. Implantation, biochemical pregnancy and ongoing pregnancy rates were similar. CONCLUSIONS: Our findings confirmed that the GnRHa triggering strategy is associated with increased number of oocytes retrieved and of mature oocytes even in normoresponder women. Moreover, in these patients, the use of intramuscular progesterone during luteal phase support achieved satisfactory IVF outcomes.

Use of the gonadotropin-releasing hormone (GnRH) stimulation test to monitor gonadal function in intact adult male cats.

The gonadotropin-releasing hormone (GnRH) stimulation test is a common procedure used to investigate normality of the pituitary-gonadal axis in mammals. There is very little information on the technique, its efficacy and side effects in small animals and in particular no information for male cats. In dogs, such test is performed by intravenous (IV) administration. With cats, the number of times the animal needs to be restrained for blood sampling should be the least possible. The purpose of this study was to assess efficacy and side effects of the GnRH stimulation test in tomcats comparing the IV with the intramuscular (IM) route of administration. A GnRH stimulation test was performed in eight adult tomcats through IM or IV administration of 50 µg gonadorelin. The response of the pituitary-gonadal axis was assessed by measuring serum testosterone on blood samples collected prior to and 1 hr following treatment. When considering each single group of cats, the post-stimulation serum testosterone values were significantly higher than the pre-treatment ones (p < .05). When comparing the two groups of cats, basal testosterone concentrations did not differ, and also post-GnRH testosterone concentrations did not differ. In conclusion, in the cats of our study, the GnRH stimulation test produced the same results following the IM or the IV route of administration. Therefore, in tomcats, the IM route can be considered as effective as the IV one and should be preferred when doing a GnRH test.

A survey of manufacturing and handling practices for monoclonal antibodies by pharmacy, nursing and medical personnel.

INTRODUCTION: There is a paucity of data available to assess the occupational health and safety risk associated with exposure to monoclonal antibodies. Industry standards and published guidelines are conflicting or outdated. Guidelines offer contrary recommendations based on an array of methodological approaches. This survey aimed to describe current practices, beliefs and attitudes relating to the handling of monoclonal antibodies by Australian medical, nursing and pharmacy clinicians. METHODS: An electronic survey was distributed between June and September 2013. Respondents were surveyed on three focus areas: institutional guideline availability and content, current practices and attitudes. Demographic data relating to respondent and primary place of practice were also collected. RESULTS: A total of 222 clinicians completed the survey, with representation from all targeted professional groups and from a variety of geographic locations. 92% of respondents reported that their institution prepared or administered monoclonal antibodies, with 87% specifically handling anti-cancer monoclonal antibodies. Monoclonal antibodies were mostly prepared onsite (84-90%) and mostly within pharmacy clean-rooms (75%) and using cytotoxic cabinets (61%). 43% of respondents reported access to institutional monoclonal antibody handling guidelines with risk reduction strategies including training and education (71%), spill and waste management (71%), procedures for transportation (57%) and restricted handling (50%). Nurses had a stronger preference towards pharmacy manufacturing than both doctors and pharmacists for a range of clinical scenarios. 95% of all respondents identified that professional or regulatory body guidelines are an important resource when considering handling practices. CONCLUSION: Monoclonal antibodies are most commonly handled according to cytotoxic drug standards and often in the absence of formal guidelines.

Australian consensus guidelines for the safe handling of monoclonal antibodies for cancer treatment by healthcare personnel.

These consensus guidelines provide recommendations for the safe handling of monoclonal antibodies. Definitive recommendations are given for the minimum safe handling requirements to protect healthcare personnel. The seven recommendations cover: (i) appropriate determinants for evaluating occupational exposure risk; (ii) occupational risk level compared with other hazardous and non-hazardous drugs; (iii) stratification of risk based on healthcare personnel factors; (iv) waste products; (v) interventions and safeguards; (vi) operational and clinical factors and (vii) handling recommendations. The seventh recommendation includes a risk assessment model and flow chart for institutions to consider and evaluate clinical and operational factors unique to individual healthcare services. These guidelines specifically evaluated monoclonal antibodies used in the Australian cancer clinical practice setting; however, the principles may be applicable to monoclonal antibodies used in non-cancer settings. The guidelines are only applicable to parenterally administered agents.

Cytokines tumor necrosis factor-α and interferon-γ participate in modulation of the equine corpus luteum as autocrine and paracrine factors.

Knowledge on the regulation of corpus luteum (CL) function in the mare is scarce. In this study, the presence of cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), and their receptors (TNFRI, TNFRII and IFNRI), was investigated in equine CL throughout the luteal phase. The effects of TNF and IFNG on secretory function and viability of luteal cells were defined in vitro. Cytokine ligands and receptors were present in steroidogenic and endothelial cells. Protein expression for TNF was greater in mid-phase and regressing CL, while TNFRI was increased in regressing CL and TNFRII did not change. IFNG and IFNRI showed the highest expression in regressing CL. Transcription of mRNA for TNF increased from mid-phase to regressing CL and both TNFRI and TNFRII decreased from early to regressing CL. Transcription of mRNA for IFNG was lower in CL from early phase than in mid or regressing luteal phase, while IFNRI expression was not changed. In the early CL, TNF acted to increase P(4) and PGE(2) but decrease PGF(2α) secretion. In the mid luteal phase, TNF increased PGF(2α) secretion and TNF+IFNG decreased PGE(2) secretion. In the regressing luteal phase, TNF, IFNG and TNF+IFNG decreased P(4) and PGE(2) secretion, but TNF and TNF+IFNG increased PGF(2α) secretion by luteal cells. Cell viability was reduced by TNF+IFNG in regressing CL. These data show the presence of cytokines TNF and IFNG, and their receptors, in the equine CL and indicate their potential involvement in regulation of luteal function.

Efficacy of two Ni-Ti systems and hand files for removing gutta-percha from root canals.

AIM: To compare the effectiveness of two Ni-Ti systems and hand files for removing gutta-percha and sealer from root canals. METHODOLOGY: The root canals of 60 single-rooted human teeth were prepared, filled with gutta-percha and sealer (Pulp Canal Sealer; SybronEndo, Orange, CA, USA). Specimens were then divided into three groups (n=20), and root filling material was removed using MTwo Retreatment Files (group 1); R-Endo (group 2); K-files and Gates-Glidden drills (group 3). After retreatment, the efficacy of each technique was assessed using radiographs that were later digitized and the images analysed using AutoCAD 2004. The percentage of residual gutta-percha was calculated for the whole canal as well as for the coronal, middle and apical thirds. Time required, apically extruded debris and the number of fractured instruments were also recorded. Data were statistically analysed using Kruskal-Wallis and Mann-Whitney U-tests. RESULTS: All instrumentation techniques left gutta-percha and sealer remnants inside the root canals. Ni-Ti systems were significantly faster (P < 0.05) than the manual technique and significantly more effective (P < 0.05) in removing gutta-percha particularly from the middle and apical thirds of the root canal. R-Endo instrumentation was significantly more effective (P < 0.05) than MTwo retreatment files in removing gutta-percha from the middle and apical thirds. R-Endo instruments were associated with the least number of cases of apical extrusion. One MTwo instrument fractured. CONCLUSIONS: The Ni-Ti systems were more effective and faster than hand files, although all techniques left gutta-percha and sealer remnants on the root canals.

Semen quality and onset of sterility following administration of a 4.7-mg deslorelin implant in adult male dogs.

The purpose of this study was to define (i) the interval between treatment and sterility, and (ii) semen quality in male dogs administered a 4.7-mg deslorelin implant. Six healthy, adult dogs of various breeds and body weights were implanted with deslorelin (Suprelorin, Virbac) and followed every 2 weeks with semen and blood collections. Semen quality remained stable or even improved during the first month following treatment and then showed a progressive decline until the end of the study, except for sperm morphology, which was unaffected by the treatment. Complete sterility was achieved on post-treatment days 70, 84, 60, 23, 51 and 40 for dogs 1 to 6, respectively. The 4.7 mg deslorelin implant caused a significant (p < 0.05) decrease in serum testosterone as well as sperm motility. Our results (i) confirm the efficacy of deslorelin in causing reversible sterility in male dogs, (ii) confirm and provide details about endocrine and seminal parameters involved in this process and (iii) contribute to define the interval between treatment and achievement of complete sterility. Practitioners should be aware that such interval may be longer than 2 months in some cases, and that fertility may actually be increased during the first 2-4 weeks post-treatment.

Who needs LH in ovarian stimulation?

LH plays a key role in the intermediate-late phases of folliculogenesis. Although ovarian stimulation is efficiently achieved in most cases by the administration of exogenous FSH alone, specific subgroups of women may benefit from LH activity supplementation during ovarian stimulation. Some authors have found improved outcome with LH activity supplementation in advanced reproductive age women. Experience suggests that in about 10-12% of young normogonadotrophic patients treated with a gonadotrophin-releasing hormone agonist (GnRH-a) long protocol plus recombinant FSH human (r-hFSH), a 'steady response' is observed. In this subgroup of women, a higher number of oocytes is retrieved when daily LH activity supplementation is given from stimulation day 8, if compared with the standard FSH dose increase. Another subgroup of patients who may benefit from LH activity supplementation are those at risk for poor ovarian response treated with GnRH antagonist. Recent data demonstrate that in these women, when GnRH is administered in a flexible protocol, the concomitant addition of recombinant human LH improves the number of mature oocytes retrieved, when compared with the standard GnRH-a flare-up protocol. Thus, well calibrated LH administration improves the ovarian outcome in patients >35 years, in those showing an initial abnormal ovarian response to r-hFSH monotherapy, and in 'low prognosis' women treated with GnRH antagonists.

Suboptimal response to GnRHa long protocol is associated with a common LH polymorphism.

The aim of this observational preliminary trial was to estimate the association between the most common polymorphism of LH (LH-ß variant: v-ßLH), with different profiles of ovarian response to recombinant human FSH (rhFSH). A total of 60 normogonadotrophic patients undergoing a gonadotrophin-releasing hormone analogue long down-regulation protocol followed by stimulation with recombinant human FSH (rhFSH) for IVF/intracytoplasmic sperm injection, and in whom at least five oocytes were retrieved were retrospectively included. On the basis of the total rhFSH consumption, patients were divided into three groups: Group A: 22 women requiring a cumulative dose of rhFSH >3500 IU; Group B: 15 patients requiring 2000-3500 IU; Group C (control): 23 women requiring <2000 IU. The presence of v-ßLH was evaluated using specific immunoassays. Peak oestradiol concentrations were significantly lower in Group A when compared with both groups B (P < 0.05) and C (P < 0.001). Group A had a significantly lower (P < 0.05) number of oocytes retrieved (7.3 ± 1.5, 11.7 ± 2.4 and 14.7 ± 4.1 in the three groups, respectively). Seven carriers (31.8%) of v-ßLH were found in Group A, whereas only one variant (6.7%) was observed in Group B; no variant was detected in Group C. These preliminary results suggest that v-ßLH is more frequent in women with ovarian resistance to rhFSH.

A huge ovarian cyst in a hysterectomized bitch.

A 11-year-old, spayed, female mixed breed-dog was presented with an abdominal mass that was detected 1 month ago. Upon abdominal palpation a large, firm, oval shaped, movable mass was found in the mid-abdominal region. Survey radiograph of the abdomen demonstrated an oval soft tissue dense mass located on the right side of the abdominal cavity. A large, heteregenous and cystic mass with solid components occupying the majority of the abdomen and a small, cystic mass with solid components caudal to the left kidney were identified by transabdominal ultrasonography. Computed tomography scans revealed bilateral ovarian masses, and a small volume of retroperitoneal fluid on the right side. A cystic, but otherwise solid mass located in the right ovary and small retained left ovary encapsulated in the ovarian bursa were excised surgically by midline laparotomy. Histopathological examination of the excised mass from the right side revealed a large cystic structure consistent with an ovarian cyst and multiple corpora lutea and follicles at different maturational stages were detected in the left ovary. The precise origin of the ovarian cyst could not be determined by morphological appearance. Immunohistochemical staining suggested a cyst of surface epithelial origin. At re-examination 6 months after the surgery, the bitch appeared healthy and the clinical findings were all normal. To our knowledge, the cyst described here is the largest reported in an incompletely ovariohysterectomized bitch.

Full recovery of subinvolution of placental sites in an American Staffordshire terrier bitch.

A three-year-old, sexually intact, female American Staffordshire terrier was presented with chronic postpartum vulvar haemorrhage. Sixty-one days before presentation, the bitch had whelped 10 puppies. A tentative diagnosis of subinvolution of placental sites was made when syncythial trophoblast-like cells were identified in the specimen collected through the opening of the cervix by endoscopy. Five weeks after the diagnosis, the bitch was clinically healthy with no vaginal bleeding and spontaneous recovery occurred without surgical treatment.

Canine adipose-derived-mesenchymal stem cells do not lose stem features after a long-term cryopreservation.

Adult stem cells are nowadays used for treating several pathologies. A putative stem cell population was found in the adipose tissue of mammals and canine adipose tissue-derived-mesenchymal stem cells (cA-MSC) have been shown to possess the capacity to differentiate into several lineages. The main goal of our research was to fully characterize cA-MSC and examine the effects of cryopreservation on their stemness features. Each sample of cA-MSC was analyzed immediately and then again after being frozen in liquid nitrogen for one year. After the cryopreservation period cells conserved their fibroblast-like morphology, alkaline phosphatase positivity and CD expression but showed a lower proliferation ratio and a lower telomerase activity in comparison with fresh cells. Finally, the cryopreservation protocol did not change the cA-MSC adipogenic, osteogenic and myogenic differentiative potential. Our data demonstrate that stored cA-MSC might represent a promising type of progenitor cell for autologous cellular-based therapies in veterinary medicine.

Pregnancy and conception rate after two intravaginal inseminations with dog semen frozen either with 5% glycerol or 5% ethylene glycol.

The primary goal of this study was to compare the effects of 5% ethylene glycol (EG) and 5% glycerol (G) on fertility of frozen-thawed dog semen following intravaginal insemination. The sperm-rich fraction of the ejaculate of three male dogs was collected, pooled and divided into two aliquots, and then frozen with a Tris-glucose-egg yolk-citric acid extender containing either 5% G or 5% EG. A total of 10 bitches were inseminated twice, five with G-frozen-thawed semen and five with EG-frozen-thawed semen; intravaginal inseminations were performed the 4th and the 5th day after the estimated LH peak; four straws, thawed in a 37 degrees C water bath for 1 min and diluted in a Tris buffer, were used for insemination (200 x 10(6) spermatozoa); the insemination dose was introduced in the cranial vagina of the bitch using a sterile plastic catheter. Ovariohysterectomy was performed in all bitches between days 29 and 31 after the calculated LH surge, and pregnancy status, and the number of conceptuses and corpora lutea were recorded. All bitches were pregnant. Neither the number of conceptuses, nor the ratio of conceptuses to corpora lutea (conception rate) was significantly different between groups. In this first screening, with a limited number of bitches, EG-frozen semen did not show a higher fertility than G-frozen semen when used for two intravaginal inseminations. Irrespective of the semen used, conception rate was 0.50.

Clinical use of deslorelin for the control of reproduction in the bitch.

This study was conducted to evaluate clinical efficacy of deslorelin for inhibiting reproduction in the bitch. Ten adult healthy bitches or bitches with mammary neoplasia for which owners were requesting suppression of cyclicity without performing gonadectomy were administered a 4.7- or a 9.4-mg deslorelin implant subcutaneously. The first implant of deslorelin was administered in anoestrus (n = 5) or in dioestrus (n = 5). Treatment was repeated every 5 months for as long as necessary based on the clinical situation of the dog and owner's desires. Some of the bitches implanted in anoestrus came in heat within 4-15 days after treatment, while none of the bitches implanted in dioestrus showed heat during treatment. Suppression of reproductive cyclicity was successfully achieved in 6/10 bitches for 1-4 years. No behavioural and local/general side-effects were observed in any of the treated bitches. The 4.7-mg deslorelin implant may work well for suppression of cyclicity provided that it is administered in dioestrus and at intervals of 4.5 months. A 9.4-mg implant may be more suitable for this use although its efficacy may also be shorter than 12 months. Owner compliance is an important limiting factor.

Effect of an injectable cabergoline formulation on serum prolactin (PRL) and milk secretion in early postpartum Beagle bitches.

This study was conducted in order to evaluate effects on prolactin (PRL) concentration and mammary milk secretion of an injectable cabergoline formulation administered to five lactating Beagle bitches during early postpartum (PP). Bitches were bled twice daily (from PP day 3 to PP day 12) and then daily (from PP day 13 to PP day 16) to assay serum PRL. On PP day 6, a subcutaneous (SC) injection of 0.1 ml/kg of placebo was administered. On PP day 9, a SC 0.1 ml/kg dose of injectable cabergoline was administered. All bitches were checked for milk production, using a clinical scoring in order to quantify milk expression from each teat. A circadian variation of serum PRL was evident during the 6 days of pre-treatment monitoring. The day after cabergoline injection, an 80% decrease of PRL serum concentration was observed (p < 0.05). The circadian oscillatory pattern of PRL secretion disappeared after administration of cabergoline, and PRL values remained significantly lower than in the previous days for the first 60 h following treatment (p < 0.001). Milk production was drastically reduced when comparing pre-treatment to post-treatment scores (p < 0.001). A single dose of injectable cabergoline caused a significant reduction in serum PRL concentration and a significant reduction in milk flow. The injectable formulation of cabergoline appeared to be safe and well tolerated.