Development of Postmeiotic Cells In Vitro from Spermatogonial Cells of Prepubertal Cancer Patients.

Aggressive chemotherapy in childhood often results in testicular damage and consequently jeopardizes future fertility. The presence of spermatogonial cells (SPGCs) in the testes of prepubertal cancer patient boys (PCPBs) can be used to develop future strategies for male fertility preservation. In the present study, we examined the presence of SPGCs in testes of chemotherapy-treated PCPBs and their ability to develop spermatogenesis in vitro using a three-dimensional culture system. Seven testicular biopsies were obtained from chemotherapy-treated PCPBs and one from a patient with ß-thalassemia major. Isolated testicular cells were cultured in a methylcellulose culture system (MCS)-containing StemPro enriched with growth factors for 5-15 weeks. The presence of premeiotic, meiotic, and postmeiotic cells was examined by immunofluorescence staining and/or reverse transcription-polymerase chain reaction (RT-PCR) analysis. We observed SPGCs in the examined testicular biopsies. Isolated testicular cells cultured in MCS developed into colonies and contained premeiotic, meiotic, and postmeiotic cells. Furthermore, we identified sperm-like cells that had developed from testicular cells of a PCPB. Our results demonstrate, for the first time, the presence of biologically active SPGCs in testicular biopsies of chemotherapy-treated PCPBs and their capacity to develop in vitro to different stages of spermatogenesis, including the generation of sperm-like cells. This study may open the way for new therapeutic strategies for fertility preservation of PCPBs and for azoospermic patients.

[FERTILITY PRESERVATION IN YOUNG CANCER PATIENTS - CAN WE OPTIMIZE THE PATH?]

INTRODUCTION: Advances in cancer therapy have improved the long-term survival of cancer patients. Concerns about fertility represent a major issue for young cancer patients. The emergent discipline of oncofertility, an intersection between oncology and fertility, is a new concept that describes an integrated network of clinical resources that focus on fertility preservation from both clinical and research perspectives. Patients and methods: In this article we describe our designated multidisciplinary program for fertility preservation in pediatric and young adult populations. The program is also designed to serve as a prospective platform for the evaluation of reproductive outcomes in this patient cohort. RESULTS: We have observed considerably higher referral rates following launching the program and earlier referral of chemonaïve patients that concedes maximal fertility preservation. Two hundred and thirty five patients were referred to the program over a period of 3 years. CONCLUSIONS: Our program demonstrates that multidisciplinary programs that encompass relevant specialists, skilled laboratory resources and a facilitated path that drives the process in the shortest time, maximizes the yield.

Predictors of spermatogenesis in radical orchiectomy specimen and potential implications for patients with testicular cancer.

OBJECTIVE: To assess the ability of semen analysis and other patients' characteristics to predict the presence of spermatozoa in radical orchiectomy pathological specimen, and describe potential implications for patients with azoospermia and testis cancer. DESIGN: Retrospective cohort study. SETTING: Tertiary hospital. PATIENT(S): A total of 214 consecutive patients with testicular cancer who underwent radical orchiectomy between 1997 and 2015. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Histologic slides were reviewed and the presence of mature spermatozoa was documented. Clinical, laboratory, and radiographic characteristics were recorded. Logistic regression analyses were used to identify factors associated with the presence of spermatozoa in the noninvolved ipsilateral testicular parenchyma. RESULT(S): Spermatozoa were found in the pathological specimen of 145 patients (67.8%). At multivariate analysis, increased tumor size was the only factor associated with lower rates of spermatozoa in the specimen. Mean tumor diameter was 4.06 cm, and spermatozoa were found in 83% and 49% of testes with tumor diameters <4 and ≥4 cm, respectively. Preoperative semen analysis records were available for 107 patients. Oligozoospermia, severe oligozoospermia, azoospermia, and cryptozoospermia were observed in 17 (16%), 18 (17%), 9 (8%) and 3 (3%) patients, respectively. Sperm concentration and motility were not associated with complete spermatogenesis. Seven of 12 patients (58%) with either azoospermia or cryptozoospermia had mature sperm in their pathological sections. CONCLUSION(S): Larger testicular cancers are associated with lower rates of spermatozoa in the ipsilateral testis. Given the substantial likelihood (∼60%) of spermatozoa to be present in the cancerous testis of patients with azoospermia and cryptozoospermia, concomitant oncologic testicular sperm extraction (TESE) can be considered in these selected patients.

Progesterone-to-follicle index is better correlated with in vitro fertilization cycle outcome than blood progesterone level.

OBJECTIVE: To investigate the impact of late follicular phase progesterone (P) elevation in relation to ovarian response on cycle outcome. DESIGN: Cohort study. The progesterone-to-follicle index (PFI) was calculated by dividing the blood P by the number of follicles ≥14 mm. The clinical pregnancy rate was calculated against the range of PFI values and blood P levels. SETTING: In vitro fertilization unit. PATIENT(S): A heterogenous population undergoing IVF with pituitary suppression and gonadotropin stimulation resulting in 3-15 follicles ≥14 mm and blood P≤10 nmol/L on hCG day and resulting in fresh embryo transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Association of blood P and PFI with clinical pregnancy rate. RESULT(S): Data were retrieved for 8,649 IVF cycles in normal responders. The (reverse) odd ratios for pregnancy were 1.112 (95% confidence interval [CI], 1.077-1.165) for blood P and 4.104 (95% CI, 3.188-5.284) for the PFI. Elevated P levels were associated with a lower pregnancy rate only when they reached the >93rd percentile. The PFI was inversely and linearly related to the pregnancy rate for the whole range of values. CONCLUSION(S): A late increase in P level is detrimental if it is a consequence of increased P production per follicle (high PFI) but not if it is a consequence of additional follicular recruitment. The PFI enables clinicians to differentiate these conditions.

Delayed presentation of complete pancreatic ductal transection in children: management of two cases without resection.

Pancreatic ductal injuries in children are rare, and ductal transections presenting in a delayed or subacute fashion are seldom reported. We describe two cases of traumatic pancreatic ductal transection secondary to physical abuse, both of which presented late to medical care. Both were managed successfully without pancreatic resection. Judicious application of non-resectional management can yield favorable outcomes in this subset of pediatric patients.

High yield of oocytes without an increase in circulating estradiol levels in breast cancer patients treated with follicle-stimulating hormone and aromatase inhibitor in standard gonadotropin-releasing hormone analogue protocols.

BACKGROUND: Adjuvant/neoadjuvant chemotherapy in breast cancer patients may be associated with amenorrhea and a marked reduction in ovarian reserve. OBJECTIVES: To assess the use of letrozole with follicle-stimulating hormone (FSH) in gonadotropin-releasing hormone (GnRH) analogue protocols, based on reported attempts to avoid the estradiol (E2) increase during controlled ovarian hyperstimulation for embryo cryopreservation in breast cancer patients using a combination of low dose FSH and aromatase inhibitor (letrozole) in a GnRH-antagonist protocol. METHODS: Twenty-four breast cancer patients were treated with recombinant FSH (150-450 U/day) and letrozole (5 mg/day) in a long GnRH-agonist (n=7) or GnRH-antagonist (n=17) protocol. After oocyte retrieval, insemination and/ or intracytoplasmic sperm injection was performed. The embryos were frozen. RESULTS: The average interval from surgery to oocyte retrieval was 40 days. Average duration of treatment was 9.6 days and mean peak E2 level 1342 +/- 1091 pmol/L, yielding 16.0 +/- 16.3 oocytes (range 0-82). Mean fertilization rate was 69.5 +/- 20.4% and mean number of embryos cryopreserved 10.3 +/- 9.3. More oocytes were retrieved with the long GnRH protocol, but the difference was not statistically significant (24.8 +/- 24.6 vs. 12.0 +/- 8.8 pmol/L, P = 0.07). CONCLUSIONS: As previously reported, ovarian stimulation with letrozole and FSH, in both the long GnRH-agonist and GnRH-antagonist protocols, is apparently effective in breast cancer patients and spares them exposure to high E2 levels.

High-quality embryos maintain high pregnancy rates in passive smokers but not in active smokers.

This study assesses the effect of passive and active smoking on pregnancy rates after IVF with transfer of high-quality embryos. In a cohort study, women attending the IVF unit in 2006­2007 with favourable parameters for pregnancy (<38 years; less than three IVF cycles, transfer of two highest-grade embryos) grouped by smoking status were included. The cohort included 237 patients/cycles: 42 smokers, 195 non-smokers. The clinical pregnancy rate was significantly lower in smokers (35.7% versus 55.4%,P = 0.021, OR = 0.44 (95% CI 0.22­0.89)), even after conditional stratification on covariates (passive smoker, passive or partner smoker, age group). The live-birth rate was lower in smokers (28.6% versus 42.6%), but the difference was not statistically significant(OR = 0.54 (0.26­1.11)). Among non-smokers, there was no difference in pregnancy rate by passive or partner smoking. On logistic regression, variables predicting pregnancy were age <35 years (P = 0.008, OR = 2.58 (1.2­5.2)) and non-smoking (P = 0.003,OR = 3.47 (1.51­7.98)). In conclusion, transfer of high-quality embryos does not overcome the negative effect of active smoking on pregnancy rate in IVF treatment. The endometrium is apparently involved in the mechanism underlying IVF failure in smokers.

High serum oestradiol concentrations in IVF cycles increase the risk of pregnancy complications related to abnormal placentation.

The study was designed to evaluate the isolated effect of high serum oestradiol concentration on human chorionic gonadotrophin (HCG) day in IVF cycles on endometrial receptivity and placentation. A retrospective cohort included all women attending the IVF unit in 2006 and 2007, with the best prognosis to achieve pregnancy: age (<38 years), less than three IVF cycles, transfer of two highest grade embryos and no evidence of factors known to impair implantation or that are associated with increased risk of pregnancy complications. The total included 280 patients were categorized into three groups according to their serum oestradiol concentration on HCG day: group 1, oestradiol <5000 pmol/l, group 2, oestradiol in the range 5000-10,000 pmol/l and group 3, oestradiol in the range of 10,000-15,000 pmol/l. No significant differences were found between the groups in implantation, pregnancy and abortion rates. The high oestradiol group was characterized by high rate (20.8%) of pregnancy complications related to abnormal placentation--fetal growth restriction, pregnancy-induced hypertension and abnormal implantation of the placenta. Hence, the decision to perform embryo transfer in high-responder patients should take into consideration both possible risks of ovarian hyperstimulation syndrome and pregnancy complications related to abnormal placentation.

Effect of coasting on IVF cycle characteristics and outcome in short vs. long GnRH agonist protocols.

AIMS: To compare the results of IVF cycles following coasting in patients treated with long versus short GnRH agonist protocols. METHODS: A retrospective comparative study in which all women aged 35 years or less attending the IVF unit from 2000 to 2006 in whom coasting was used in GnRH agonist protocols were included. Data on coasting-related variables and outcome were collected from the files and compared between the short GnRH agonist (n = 78) and long GnRH agonist (n = 181) cycles. RESULTS: The short GnRH agonist cycles were characterized by higher E2 levels during coasting and longer duration of coasting than the long GnRH agonist cycles. Although the number of retrieved oocytes was lower following coasting in the short protocol, there was no difference between the groups in fertilization rate, number of high-quality embryos available for transfer, and pregnancy rate. Pregnancy rate in both protocols was negatively correlated to E2 level at initiation of coasting. The overall moderate and severe OHSS rate after coasting was 5.1% in the short-protocol group and 6.0% in the long-protocol group (p = 0.76). CONCLUSIONS: The ovarian response curve to coasting is longer in the short than in the long GnRH-agonist protocol, but there is no significant difference in pregnancy or OHSS rates.

In vitro fertilization cycle outcome after coasting in gonadotropin-releasing hormone (GnRH) agonist versus GnRH antagonist protocols.

OBJECTIVES: To compare the results of IVF cycles after coasting in patients treated with a GnRH antagonist or GnRH agonist protocol. DESIGN: A retrospective case-control study. SETTING: Infertility unit in a university-affiliated tertiary medical center. PATIENT(S): The study group included all women less than 38 years old attending the IVF unit from 2000 to 2006 in whom coasting was used. Data on E(2) levels before and after coasting, duration of coasting, number of oocytes retrieved and fertilized, embryo quality, moderate-severe ovarian hyperstimulation syndrome (OHSS), and pregnancy were collected from the files and compared between GnRH agonist (n = 329) and GnRH antagonist (n = 45) cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Number of retrieved oocytes and pregnancy rates. RESULT(S): There were no between-group differences in cycle parameters. In the antagonist group, there was no need for more than 2 days of coasting. There was a significant decrease in the number of retrieved oocytes even in short periods of coasting in the antagonist group but not in the agonist group. On the day of hCG administration, E(2) levels dropped to a lower level in the antagonist cycles. The OHSS rate after coasting was 4.6% in the agonist group and 4.4% in the antagonist group. Corresponding pregnancy rates after coasting were 27.4% and 24.4%. CONCLUSION(S): The same criteria for coasting can be applied in GnRH agonist as in GnRH antagonist cycles, with a similar IVF result.

Biliary leaks after laparoscopic cholecystectomy: time to stent or time to drain.

BACKGROUND: Endoscopic retrograde cholangiopan-creatography (ERCP) with placement of a biliary stent or nasobiliary (NB) drain is the procedure of choice for treatment of post-cholecystectomy bile duct leaks. The aim of this study was to compare the effect of NB drainage versus internal biliary stenting on rates of leak closure, time elapsed until drain or stent removal, length of hospital stay and number of required endoscopic procedures. METHODS: Charts were reviewed on 20 patients who underwent laparoscopic cholecystectomy complicated by Luschka or cystic duct leak. Ten patients were treated with NB drains connected to low intermittent suction and repeat NB cholangiograms were performed until leak closure was observed. Ten patients were treated with internal biliary stents. Biliary sphincterotomies were performed for stone extraction or a presumed papillary stenosis. Large bilomas were drained percutaneously prior to stenting. RESULTS: In all 20 patients, a cholangiogram and successful placement of a NB drain or internal stent was achieved. Four patients (20%) were found to have bile duct stones, which were extracted following a sphincterotomy. Sixteen patients required percutaneous drains to evacuate large bilomas prior to biliary instrumentation. Fifteen cystic duct leaks and 5 Luschka duct leaks were reviewed. There were no complications related to ERCP. Closure of the leak was documented within 2 to 11 days (mean 4.7+/-0.9 days) in patients receiving a NB drain. The drains were removed non-endoscopically following leak closure. The internal stent group required stenting for 14 to 53 days (mean 29.1+/-4.4 days). The stent was then removed endoscopically after documentation of leak closure. Bile leaks following laparoscopic cholecystectomy closed rapidly after NB drainage and did not require repeat endoscopy for removal of the NB drain, resulting in fewer ERCPs required for treatment of biliary leaks. Internal biliary stents were in place longer owing to the nature of this intermittent endoscopic approach but an accurate comparison of time to leak closure could not be determined. Leak closure resulted once the bile flow was re-established, regardless of the technique, but removal of the NB drains was performed earlier than removal of the biliary stents. The number of ERCPs required per patient was 1.0+/-0 in the NB group and 2.2+/-0.1 (range 2-3) in the internal stent group. The length of hospitalization was 8.7+/-3.3 days for the NB group and 7.5+/-2.3 days for the internal stent group. Biliary stent placement resulted in an insignificant decrease in hospitalization at the expense of generating twice as many endoscopic procedures. CONCLUSIONS: Our data suggest that NB drainage may be advantageous in patients requiring a prolonged hospital admission or in patients in whom repeat endoscopy is undesirable. Internal biliary stenting appears preferable when early discharge is anticipated or when expertise in placement and management of NB drains is lacking.

The effect of oral contraceptive pill for cycle scheduling prior to GnRH-antagonist protocol on IVF cycle parameters and pregnancy outcome.

OBJECTIVE: To evaluate the effect of oral contraceptive pills (OCP) pretreatment on IVF cycle outcome in GnRH-antagonist protocol. DESIGN: Retrospective cohort study. SETTING: Major tertiary university-affiliated center. PATIENTS: All patients treated with GnRH antagonist in our IVF unit during the last 3 years were included in the study. Overall 1,799 IVF cycles were performed. Of these, in 604 cycles OCP pretreatment was used prior to GnRH-antagonist for cycle scheduling. Patients were divided into two age groups-young group aged < or = 35 years and older group aged > or = 36 years. INTERVENTIONS: The young group underwent 927 cycles, 281 cycles with OCP pretreatment and 646 cycles without. The older group underwent 872 cycles, 323 cycles with OCP pretreatment and 549 cycles without. Data was analyzed within each age group. MAIN OUTCOME MEASURES: Treatment duration and total dose of FSH IU used for stimulation, number of oocytes retrieved, implantation and pregnancy rates. RESULTS: All OCP-pretreated cycles required significantly longer stimulation than non-pretreated cycles (young: 10.76 vs. 9.21 days; older: 10.48 vs. 8.73 days, respectively) and higher total dose of FSH IU (young: 3,210 IU vs. 2,565 IU; older: 4,973 IU vs. 3,983 IU, respectively). There were no other differences in cycle characteristics between groups. Implantation and pregnancy rates were not affected by OCP pretreatment. CONCLUSIONS: OCP pretreatment can be offered as a mode for cycle scheduling prior to GnRH-antagonist protocol, though it may be associated with longer stimulation and higher gonadotropin consumption.

The importance of routine liver biopsy in diagnosing nonalcoholic steatohepatitis in bariatric patients.

BACKGROUND: Nonalcoholic Steatohepatitis (NASH) commonly occurs in obese patients and predisposes to cirrhosis. Prevalence of NASH in bariatric patients is unknown. Our aim was to determine the role of routine liver biopsy in managing bariatric patients. METHODS: Prospective data on patients undergoing Roux-en-Y gastric bypass (RYGBP) was analyzed. One pathologist graded all liver biopsies as mild, moderate or severe steatohepatitis. NASH was defined as steatohepatitis without alcoholic or viral hepatitis. Consecutive liver biopsies were compared to those liver biopsies selected because of grossly fatty livers. RESULTS: 242 patients underwent open and laparoscopic RYGBP from 1998-2001. Routine liver biopsies (68 consecutive patients) and selective liver biopsies (additional 86/174, 49%) were obtained. Findings of cirrhosis on frozen section changed the operation from a distal to a proximal RYGBP. The two groups were similar in age, gender, and BMI. The group with the routine liver biopsies showed a statistically significant larger preponderance of NASH (37% vs 32%). Both groups had a similar prevalence of cirrhosis. Neither BMI nor liver enzymes predicted the presence or severity of NASH. CONCLUSIONS: Routine liver biopsy documented significant liver abnormalities in a larger group of patients compared with selective liver biopsies, thereby suggesting that liver appearance is not predictive of NASH. Liver biopsy remains the gold-standard for diagnosing NASH. We recommend routine liver biopsy during bariatric operations to determine the prevalence and natural history of NASH, which will have important implications in directing future therapeutics for obese patients with NASH and for patients undergoing bariatric procedures.

Glutathione-S-transferase pi expression and activity is increased in colonic neoplasia.

Glutathione-S-transferase (GST) isoenzymes are involved in the conjugation of glutathione to electrophilic carcinogens. Recent studies have shown increased levels and activities of GST in different tumors suggesting their role in carcinogen detoxification. This study compared GST activity levels and GST-pi protein expression in paired samples of colorectal cancer, adenoma and adjacent normal mucosa from a total of thirteen patients. GST was isolated from human colorectal specimens and assayed spectrophotometrically; Western immunoblot analysis was used to quantify GST-pi levels. GST activity was greater in both colorectal cancer and adenomas than in adjacent normal colonic tissue, although statistical significance was achieved only when comparing colorectal cancer to normal tissue. Based on these observations, we conclude that increased GST activity may be a useful marker of colonic neoplasia.