Safety of ovarian cryopreservation and transplantation in patients with acute leukemia: a case series.

BACKGROUND: With increased success, ovarian tissue cryopreservation has recently become a standard technique for fertility preservation. However, malignant cell introduction through ovarian tissue transplantation remains a major concern for patients with acute leukemias. OBJECTIVE: This study aimed to investigate the safety of performing autologous ovarian tissue transplantation in survivors of acute leukemia. STUDY DESIGN: Clinical, histopathological, and molecular data of 4 women with acute myeloid leukemia and 2 women with acute lymphoblastic leukemia who underwent ovarian tissue cryopreservation and transplantation were analyzed in this case series. Following cryopreservation of 66% to 100% of an ovarian cortex with a slow freezing method, all women received high-dose multiagent alkylating preconditioning chemotherapy for allogeneic hematopoietic stem cell transplantation. Before the ovarian tissue transplantation, (1) antral follicle counts, serum antimüllerian hormone and follicle-stimulating hormone levels were assessed to confirm primary ovarian insufficiency; (2) all recipients were cleared by their hematologist-oncologists; (3) representative cortical strips were screened for leukemia infiltration by histologic (hematoxylin and eosin staining), immunohistochemical (CD3, CD20, CD34, CD68, CD117, CD163, PAX-5, Tdt, lysozyme, and MPO), and molecular marker evaluation (BCR/ABL p190 and AML1/ETO) where appropriate. RESULTS: The median age was 20 years (interquartile range, 15-32) at ovarian tissue cryopreservation. Before undergoing hematopoietic stem cell transplantation, all patients received induction or consolidation chemotherapy that included cytarabine + daunorubicin or Berlin-Frankfurt-Munich-95 protocol and were in remission. The mean serum antimüllerian hormone was 1.9±1.7 ng/mL before ovarian tissue cryopreservation. In all cases, ovarian tissue screening for leukemic cells was negative. Ovarian transplantation was performed laparoscopically with or without robotic assistance, after a median of 74.5 months (interquartile range, 41-120) after ovarian tissue cryopreservation. Ovarian function resumed in all patients after a median of 3.0 months (range, 2.5-4.0), and 2 women had 1 live birth each. The median graft longevity was 35.5 months (interquartile range, 18-57) after ovarian tissue transplantation. After a median follow-up of 51 months (interquartile range, 20-74), all patients remained relapse-free. In 1 patient, the graft was removed during cesarean delivery and was negative for immunochemical leukemia markers. CONCLUSION: Our long-term follow-up demonstrated no evidence of disease relapse after ovarian tissue transplantation in patients with acute leukemia who received allogeneic hematopoietic stem cell transplantation. This safety profile may be explained by the fact that these patients are induced into remission by nongonadotoxic induction chemotherapy before undergoing ovarian tissue cryopreservation. We propose that ovarian tissue cryopreservation should not be excluded as a fertility preservation option for young women with leukemia who are due to receive preconditioning chemotherapy before allogeneic hematopoietic stem cell transplantation.

The effects of coenzyme Q10 and curcumin supplementation in freezing medium for human sperm cryopreservation.

BACKGROUND: Despite its routine and frequent application, cryopreservation of human sperm is far from the desired efficacy, as freezing and thawing impair motility, viability, acrosomal unity, and DNA integrity. OBJECTIVES: In this study, the authors aimed to investigate whether adding antioxidants, coenzyme Q10, and curcumin into the freezing medium provide better efficacy in the cryopreservation of human sperm. METHODS: The semen samples from 40 healthy men aged 18-45 were collected in sterile containers by masturbation. Samples within normal reference values for sperm concentration (≥15 million/mL) and motility (progressive motile ≥ 32% and total motility ≥ 40%) were included in the study. Semen samples were equally divided into five groups and evaluated; i) pre-freezing sperm suspension, ii) frozen-thawed control (Ctrl) without any supplementation in freezing medium, iii) frozen-thawed with curcumin supplementation of 0.25 mM (Cur), iv) frozen-thawed coenzyme Q10 supplementation of 25 µM (CoQ10) and v) frozen-thawed curcumin (0.25 mM) plus coenzyme Q10 (25 µM) supplementation (CurCoQ10) into the freezing medium. Liquid nitrogen vapour freezing and rapid thawing were performed in each group (ii-v). Sperm motility, viability, acrosome integrity, and DNA fragmentation rates were compared and ultrastructural evaluations by transmission electron microscopy were undertaken between the groups. Additionally, the total antioxidant capacity/total oxidant capacity values were measured. RESULTS: According to CASA results, progressive motility was significantly higher in the CoQ10 group (9.4 ± 7.6) when compared with the Ctrl (7.1 ± 6.3), Cur (6.4 ± 4.8) and CurCoQ10 (8.1 ± 7.7) groups (p < 0.05). Flow cytometry results showed no difference in the viability and acrosome integrity values after thawing, but DNA fragmentation was significantly increased in the curcumin-added groups (p < 0.05). Acrosomal changes and sub-acrosomal defects were seen in all groups after thawing at the ultrastructural level. Mitochondrial membrane structure was preserved in CoQ10 and CurCoQ10 groups. CONCLUSIONS: Our results suggested that sperm ultrastructural morphology and motility were better preserved in the CoQ10 group during cryopreservation. In curcumin groups, DNA fragmentation and head defects were increased.

Recombinant anti-Mullerian hormone treatment attenuates primordial follicle loss after ovarian cryopreservation and transplantation.

PURPOSE: The foremost drawback of ovarian tissue cryopreservation and re-transplantation (OTCT) technique is the rapid loss of the primordial follicle (PF) pool. In recent studies, we have demonstrated that post-transplantation burnout of the PFs occurs due to the altered expression of the activatory and inhibitory proteins that control PF reserve, and rapamycin prevented it. METHODS: Here, we investigated whether anti-Mullerian hormone administration in the bilateral oophorectomy and transplantation group and internal AMH in the unilateral oophorectomy and transplantation group protect follicle reserve by regulating the expression of the molecules that control follicle growth after OTCT in mice. RESULTS: After 14 days of OTCT, PF reserve is significantly reduced in both unilateral oophorectomy and transplantation and bilateral oophorectomy and transplantation groups, while anti-Mullerian hormone treatment attenuates PF loss after bilateral oophorectomy and transplantation. The expression of KitL, Bmp-15, and p27 decreased after unilateral oophorectomy and transplantation and bilateral oophorectomy and transplantation, yet recombinant anti-Mullerian hormone treatment did not restore the expression of these proteins in the BLO-T group. CONCLUSION: Exogenous recombinant anti-Mullerian hormone administration in the BLO-T group preserved the expressions of Tsc1 and Gdf-9 in PF and p-s6k and Gdf-9 in growing follicles after OTCT. Nonetheless, recombinant anti-Mullerian hormone administration did not affect granulosa cell proliferation and death rates in the growing follicles. These findings suggest a novel hormonal replacement strategy for fertility preservation by restoring anti-Mullerian hormone to regulate Tsc1 and p-s6k, thereby linking this hormone with the mTOR pathway and Gdf-9 signaling.

Co-Culture of Cryopreserved Healthy Sertoli Cells with Testicular Tissue of Non-Obstructive Azoospermia (NOA) Patients in Culture Media Containing Follicle-Stimulating Hormone (FSH)/Testosterone Has No Advantage in Germ Cell Maturation.

Different cell culture conditions and techniques have been used to mature spermatogenic cells to increase the success of in vitro fertilization. Sertoli cells (SCs) are essential in maintaining spermatogenesis and FSH stimulation exerts its effect through direct or indirect actions on SCs. The effectiveness of FSH and testosterone added to the co-culture has been demonstrated in other studies to provide microenvironment conditions of the testicular niche and to contribute to the maturation and meiotic progression of spermatogonial stem cells (SSCs). In the present study, we investigated whether co-culture of healthy SCs with the patient's testicular tissue in the medium supplemented with FSH/testosterone provides an advantage in the differentiation and maturation of germ cells in NOA cases (N = 34). In men with obstructive azoospermia (N = 12), healthy SCs from testicular biopsies were identified and purified, then cryopreserved. The characterization of healthy SCs was done by flow cytometry (FC) and immunohistochemistry using antibodies specific for GATA4 and vimentin. FITC-conjugated annexin V/PI staining and the MTT assay were performed to compare the viability and proliferation of SCs before and after freezing. In annexin V staining, no difference was found in percentages of live and apoptotic SCs, and MTT showed that cryopreservation did not inhibit SC proliferation compared to the pre-freezing state. Then, tissue samples from NOA patients were processed in two separate environments containing FSH/testosterone and FSH/testosterone plus co-culture with thawed healthy SCs for 7 days. FC was used to measure 7th-day levels of specific markers expressed in spermatogonia (VASA), meiotic cells (CREM), and post-meiotic cells (protamine-2 and acrosin). VASA and acrosin basal levels were found to be lower in infertile patients compared to the OA group (8.2% vs. 30.6% and 12.8% vs. 30.5%, respectively; p < 0.05). Compared to pre-treatment measurements, on the 7th day in the FSH/testosterone environment, CREM levels increased by 58.8% and acrosin levels increased by 195.5% (p < 0.05). Similarly, in medium co-culture with healthy SCs, by day 7, CREM and acrosin levels increased to 92.2% and 204.8%, respectively (p < 0.05). Although VASA and protamine levels increased in both groups, they did not reach a significant level. No significant difference was found between the day 7 increase rates of CREM, VASA, acrosin and protamine-2 in either FSH/testosterone-containing medium or in medium additionally co-cultured with healthy SCs (58.8% vs. 92.2%, 120.6% vs. 79.4%, 195.5% vs. 204.8%, and 232.3% vs. 198.4%, respectively; p > 0.05). Our results suggest that the presence of the patient's own SCs for maturation of germ cells in the culture medium supplemented with FSH and testosterone is sufficient, and co-culture with healthy SCs does not have an additional advantage. In addition, the freezing-thawing process would not impair the viability and proliferation of SCs.

Transabdominal ultrasound-guided oocyte retrieval for oocyte cryopreservation using a vaginal probe: a comparison of applicability, effectiveness, and safety with conventional transvaginal approach.

PURPOSE: We aimed to compare the feasibility, effectiveness, and safety of transabdominal ultrasound-guided oocyte retrieval (TUGOR) using a vaginal probe and traditional vaginal approach in virgin patients undergoing oocyte cryopreservation. METHODS: A total of 116 virgin patients who underwent transabdominal ultrasound-guided oocyte retrieval using a vaginal ultrasound probe and 33 patients matched for BMI, antral follicle count, age, day 3 FSH, estradiol, and AMH who underwent vaginal approach were enrolled. Mean number of total oocytes collected, mean number of cryopreserved MII oocytes, duration of the procedure, duration of stimulation, mean gonadotropin consumption, mature oocyte ratio, and a modified follicle-oocyte index were compared between the groups. RESULTS: No statistical difference was found between the groups in mean number of follicles > 12 mm (4.62 ± 4.54 vs. 5.44 ± 4.52), mean number of oocytes collected (4.44 ± 4.14 vs. 5.33 ± 4.52), mean number of cryopreserved MII oocytes (4.01 ± 3.67 vs. 4.53 ± 4.13), mean duration of the procedure (12.4 ± 1.2 vs. 13.4 ± 1.6 min), mean days of stimulation (8.05 ± 1.91 vs. 8.35 ± 1.72 days), mean gonadotropin consumption (1507.9 ± 475.3 vs. 1571.74 ± 404.6 units), mature oocyte ratio (0.78 ± 0.24 vs. 0.82 ± 0.26), and modified follicle oocyte index (0.86 ± 0.63 vs. 0.84 ± 0.19). In the TUGOR group, superficial epigastric artery injury occurred in two patients and resolved spontaneously. CONCLUSION: Transabdominal oocyte retrieval using a vaginal ultrasound is a safe, effective, and feasible method of oocyte retrieval in some selected patient groups.

Phospholipase C-zeta levels are not correlated with fertilisation rates in infertile couples.

In mammals, 'oocyte activation' is triggered by certain proteins, one of which is phospholipase C-zeta. Recent evidence suggests that low expression of phospholipase C-zeta might be associated with male infertility, while a limited number of studies claimed the opposite. This study was designed to test whether quantity of phospholipase C-zeta and in vitro fertilisation rates are correlated or not, assessed by flow cytometry. Semen samples from 43 infertile couples were analysed for the percentage and mean fluorescent intensity (MFI) of phospholipase C-zeta protein. Results were confirmed by immunofluorescent labelling. Patients with a fertilisation rate of 40% or lower were involved in the low fertilisation group, while the high fertilization group consisted of patients with a fertilisation rate of 60% and higher. Quantitative analyses by flow cytometry showed no significant difference among the low fertilisation and high fertilisation groups when phospholipase C-zeta ratio or MFI was considered. No correlation was found between pregnancy rates and phospholipase C-zeta quantity. None of the total fertilisation failure cases were lack of phospholipase C-zeta. In fact, fertilisation was possible even when phospholipase C-zeta levels were very low. Thus, we concluded that phospholipase C-zeta quantity cannot be considered as a diagnostic tool for male infertility.

Current and Future Perspectives for Improving Ovarian Tissue Cryopreservation and Transplantation Outcomes for Cancer Patients.

Although advances in cancer treatment and early diagnosis have significantly improved cancer survival rates, cancer therapies can cause serious side effects, including ovarian failure and infertility, in women of reproductive age. Infertility following cancer treatment can have significant adverse effects on the quality of life. However, established methods for fertility preservation, including embryo or oocyte cryopreservation, are not always suitable for female cancer patients because of complicated individual conditions and treatment methods. Ovarian tissue cryopreservation and transplantation is a promising option for fertility preservation in pre-pubertal girls and adult patients with cancer who require immediate treatment, or who are not eligible to undergo ovarian stimulation. This review introduces various methods and strategies to improve ovarian tissue cryopreservation and transplantation outcomes, to help patients and clinicians choose the best option when considering the potential complexity of a patient's situation. Effective multidisciplinary oncofertility strategies, involving the inclusion of a highly skilled and experienced oncofertility team that considers cryopreservation methods, thawing processes and devices, surgical procedures for transplantation, and advances in technologies, are necessary to provide high-quality care to a cancer patient.

Efficacy and safety of papaverine as an in vitro motility enhancer on human spermatozoa.

PURPOSE: The aim of this study was to examine the ability and safety of papaverine supplementation for in vitro sperm motility enhancement. In addition, sperm motility enhancement of papaverine was compared to pentoxifylline and theophylline. The post-thaw spermatozoa were used as an asthenozoospermia model. METHODS: Post thaw sperm suspensions were divided into two groups: papaverine (100 µmol/L) and control, and each was investigated in two subgroups of 30- and 60-min exposure times. Detailed motility parameters were detected using a computerized sperm motility analyzer. Acrosomal status, viability, apoptosis, and DNA fragmentation were evaluated by flow cytometry. Furthermore, the motility-enhancing capacity of papaverine, pentoxifylline, and theophylline was compared. RESULTS: Cryopreservation impaired sperm parameters dramatically but no significant changes occurred in acrosomal status and apoptosis. Supplementation of papaverine enhanced motility parameters consistently at all exposure intervals, significantly. However, viability was lower at the 60th minute compared to the 30th minute (p=0.019). Papaverine did not alter any acrosomal or apoptotic markers at any time points. All of the compounds compared in this study increased the motility parameters, where theophylline supplementation provided significantly better improvement in total motility compared to papaverine and pentoxifylline. CONCLUSION: Our results suggest that in vitro papaverine treatment for 30 min adequately improves motility of post-thaw sperm, without leading to acrosome reaction, DNA damage, and viability loss. Theophylline's potency on increasing the ratio of total motile spermatozoa was found significantly superior than the two tested compounds. Prospective clinical studies with embryo production, pregnancy, and live birth data should be undertaken.

Deleterious reproductive effects of nilotinib in mouse model.

Nilotinib is a second-generation tyrosine kinase inhibitor (TKI) that is widely used to treat patients with Philadelphia chromosome-positive chronic myeloid leukaemia (CML). TKIs provided a significant improvement in terms of survival rates and disease-free period in CML; however, there is insufficient knowledge about their side effects, including reproductive toxicity. Since nearly half of the CML patients are in their reproductive age, and newly announced indications cover the treatment of the paediatric age groups, concerns arise about the effects of these drugs on the reproductive system, as there are no controlled preclinical studies. We investigated acute and long-term gonadotoxic and teratogenic effects of nilotinib, utilising a mouse model that simulates various clinical scenarios. We observed significant testicular damage in mice receiving nilotinib according to Johnsen's score analysis. Alterations were observed in female mice's number of follicles, as the primordial follicle numbers significantly decreased. Proliferating cell number in both genders' gonads decreased and apoptosis rate increased significantly. The nilotinib-received female and male mice's pregnancy rates were low compared to controls. A significant decrease in the thickness of the spongiotrophoblast and decidual layers of the placenta was detected in pregnancies consisting of male and/or female mice treated with nilotinib. The results of this study establish a critical point of view for clinical translation and indicate the importance of consulting patients for directing them to fertility preservation and contraception options for both genders before nilotinib treatment.

Comparison of Oocyte and Embryo Quality Between Random Start and Controlled Ovarian Stimulation Cycles in Cancer Patients Undergoing Fertility Preservation.

Conventional assisted reproductive technology (ART) cycles may delay cancer treatment and compromise survival, and also increase patients' psychological burden as a result of delayed chemotherapy. The aim of this study was to compare the success rates of random start and conventional start GnRH antagonist protocols in terms of oocyte and embryo outputs in cancer patients. Data of 111 patients with a newly diagnosed cancer who underwent ART for fertility preservation at a university-based infertility clinic between January 2010 and September 2019 were reviewed. The study group underwent random start controlled ovarian hyperstimulation (RS-COH) and the control group underwent conventional start COH (CS-COH). The main outcome measures were the number of total oocytes, MII oocytes, and embryo yield. A total of 46 patients (41.5%) underwent RS-COH and 65 (58.5%) underwent CS-COH. Baseline characteristics were similar between the groups. The most common cancer type in both groups was breast cancer (60.9% vs. 52.3%, respectively). The median duration of stimulation was significantly longer in RS-COH than in CS-COH (12 vs. 10 days; P = 0.005). The median number of MII oocytes was significantly higher in RS-COH than in CS-COH (7 vs. 5 oocytes, respectively; P = 0.020). The MII/AFC ratio was significantly higher in the RS-COH group compared to the CS-COH group (74% and 57% respectively; p = 0.02). In the linear regression analyses, RS-COH protocol did not have a significant impact on MII/AFC (standardized ß coefficient - 0.514; P = 0.289 {adjusted R2 for the model = 0.779}), oocyte yield (standardized ß coefficient - 0.070; P = 0.829 {adjusted R2 for the model = 0.840}), and MII rate (standardized ß coefficient - 0.504; P = 0.596 {adjusted R2 for the model = 0.271}). In conclusion, RS-COH protocol is as effective as CS-COH protocols for fertility preservation in cancer patients.

Leptin promotes proliferation of neonatal mouse stem/progenitor spermatogonia.

PURPOSE: To keep and increase spermatogonial stem cell number (SSC) is the only available option for pediatric cancer survivors to maintain fertility. Leptin is secreted by the epididymal white adipose tissue and has receptors on stem/progenitor spermatogonia. The purpose of this study is to demonstrate dose- and time-dependent proliferative effect of leptin on stem/progenitor spermatogonia cultures from prepubertal mice testes. METHODS: CD90.2 (+) stem/progenitor spermatogonia were isolated from the C57BL/6 mouse testis on postnatal day 6 and placed in culture. The proliferative effect of leptin supplementation was assessed by colony formation (diameter and number), WST proliferation assays, and xCELLigence real-time cell analysis (RTCA) on days 3, 5, and 7 of culture. Expressions of p-ERK1/2, p-STAT3, total STAT3, and p-SHP2 levels were determined by western blot analysis. RESULTS: Leptin supplementation of 100 ng/ml increased the diameter (p = 0.001) and number (p = 0.01) of colonies in stem/progenitor spermatogonial cultures and caused higher proliferation by WST-1 (p = 0.009) compared with the control on day 7. The EC50 was calculated as 114 ng/ml for leptin by RTCA. Proliferative dose of leptin induced increased expression of p-ERK1/2 (p = 0.009) and p-STAT3 (p = 0.023) on stem/progenitor spermatogonia when compared with the untreated group. CONCLUSION: The results indicated that leptin supplementation exhibited a dose- and time-dependent proliferative effect on stem/progenitor spermatogonia that was associated with increased expression of ERK1/2 and STAT3 pathways while maintaining their undifferentiated state. This output presents a new agent that may help to expand the stem/progenitor spermatogonia pool from the neonatal testis in order to autotransplant after cancer treatment.

Altered expression of activator proteins that control follicle reserve after ovarian tissue cryopreservation/transplantation and primordial follicle loss prevention by rapamycin.

PURPOSE: We investigated whether expression of activator proteins that control follicle reserve and growth change after ovarian tissue vitrification and re-transplantation. Moreover, we assessed whether inhibition of mTOR signaling pathway by rapamycin would protect primordial follicle reserve after ovarian tissue freezing/thawing and re-transplantation. METHODS: Fresh control, frozen/thawed, fresh-transplanted, frozen/thawed and transplanted, rapamycin/control, rapamycin fresh-transplanted, and rapamycin frozen-thawed and transplanted groups were established in rats. After freezing and thawing process, two ovaries were transplanted into the back muscle of the same rat. After 2 weeks, grafts were harvested, fixed, and embedded into paraffin block. Normal and atretic primordial/growing follicle count was performed in all groups. Ovarian tissues were evaluated for the dynamic expressions of Gdf-9, Bmp-15, KitL, Lif, Fgf-2, and p-s6K using immunohistochemistry, and H-score analyses were done. RESULTS: Primordial follicle reserve reduced almost 50% after ovarian tissue re-transplantation. Expression of Gdf-9 and Lif increased significantly in primordial and growing follicles in frozen-thawed, fresh-transplanted, and frozen/thawed and transplanted groups, whereas expression of Bmp-15, KitL, and Fgf-2 decreased in primordial follicles. Freezing and thawing of ovarian tissue solely significantly increased p-s6K expression in primordial follicles, and on the other hand, suppression of mTORC1 pathway using rapamycin preserved the primordial follicle pool. CONCLUSION: Altered expressions of activator proteins that regulate primordial follicle reserve and growth may lead to primordial follicle loss and rapamycin treatment can protect ovarian reserve after ovarian tissue cryopreservation/transplantation.

First pregnancy and live birth in Turkey following frozen-thawed ovarian tissue transplantation in a patient with acute lymphoblastic leukemia who underwent cord blood transplantation.

PURPOSE: To report the first live birth after frozen-thawed ovarian transplantation in Turkey and the second case for an acute lymphoblastic leukemia (ALL) survivor in the world. METHODS: A 19-year-old patient underwent ovarian tissue cryopreservation (OTC) before cord blood transplantation in 2010. She was diagnosed as ALL with a bone marrow biopsy revealing 90% blast ALL-L2 type, and karyotype analyses indicated reciprocal translocation at t(9;22)(q34;q11). The patient received the Berlin-Frankfurt-Munster (BFM) protocol, and complete remission was achieved before fertility preservation. Serum AMH level was measured as 1.5 ng/ml, and 12 antral follicles were counted on ultrasound. She was informed about fertility preservation options and decided to proceed with OTC, with her signed consent before cord blood transplantation in April 2011. Ovarian tissue transplantation (OTT) was performed in 2017 when the patient was menopausal with serum FSH levels > 100 IU/ml and estradiol < 20 pg/ml and hematologically in molecular remission. Detailed molecular analysis, standard histology, and immunohistochemistry demonstrated that the thawed tissue is free of malignant cells. RESULTS: Six months following OTT, she had spontaneous menstruation with serum FSH 11 IU/ml and estradiol 53 pg/ml. Two consecutive IVF cycles yielded three top-quality embryos. Following three embryo transfer cycles, one fresh and two frozen, a healthy term live birth was achieved. Frozen-thawed-transplanted tissues were extracted during caesarean delivery upon the patient's request after a total period of 25 months in vivo, and histopathological evaluation revealed that the tissue was free of leukemic infiltration. CONCLUSION: The authors report the first pregnancy and live birth in Turkey and the second live birth in the world following transplantation of frozen-thawed ovarian tissue in a leukemia survivor. As the transplanted tissues were removed during caesarean delivery, histological findings prove the functionality and the malignant-free status of the transplanted tissue during the grafted period.

Evaluation of blood-testis barrier integrity in terms of adhesion molecules in nonobstructive azoospermia.

Blood-testis barrier (BTB) is critical for maintaining fertility. The integrity of tight junctions (TJs) provides restricted permeability of BTB. The aim of this study was to evaluate the relationship between BTB and Sertoli cells. Testicular sperm extraction (TESE) obtained from nonobstructive azoospermia (NOA) patients was examined: Group I (spermatozoa+) and Group II (spermatozoa-). The tissues were stained with haematoxylin eosin, periodic acid-Schiff and Masson's trichrome for Johnsen's score evaluation. Apoptosis and adhesion molecules such as claudin-11, occludin and ZO-1 were assessed. In Group I, the integrity of the seminiferous tubules was intact. In Group II, some seminiferous tubule walls were lined only with Sertoli cells, had a thickening of the basement membrane, and oedema in interstitial spaces. In Group I, the seminiferous tubule consisted of a stratified columnar epithelium, claudin-11 expressions were observed as linear staining in the basal zone of the tubule, while seminiferous tubules, with low epithelium, displayed a punctate type of staining. Immunohistochemical observations were consistent with the ultrastructural findings. In Group II, high apoptosis and unstained/irregular TJ formation in claudin-11, occludin and ZO-1 were observed. In conclusion, disruption of relation between BTB and TJs may reveal inadequate spermatogenesis, which is one of the mechanisms behind azoospermia.

PFA is superior to glyoxal in preserving oocyte, embryo, and stem cell proteins evidenced by super-resolution microscopical surveys of epitopes.

PURPOSE: Chemical fixation is a critical step to retaining cellular targets as naturally as possible. Recent developments in microscopy allow sophisticated detection and measuring techniques with which spatio-temporal molecular alterations are conceivable. In this study, we compare two members of aldehyde fixatives [i.e., glyoxal (Gly) and paraformaldehyde (PFA)] to determine whether Gly, a less toxic dialdehyde fixative that is considered to retain immunoreactivity could provide a successful and consistent cell fixation in favor of PFA in various cell preparations and types. METHODS: We document the fixation competence of Gly and PFA side-by-side (with or without Triton X-100 permeabilization) in live- and fixed-cell preparations in mouse oocytes, embryos, and human somatic cells (human umbilical cord-derived mesenchymal stromal cells) using protein quantification by Western blot assay and super-resolution microscopy. RESULTS: Although Gly seemed to act faster than PFA, catastrophic consequences were found not acceptable, especially in oocytes and embryos. Due to cell lysate and immunocytochemistry surveys, it was obvious that PFA is superior to Gly in retaining cellular proteins in situ with little/no background staining. In many samples, PFA revealed more reliable and consistent results regarding the protein quantity and cellular localization corresponding to previously defined patterns in the literature. CONCLUSION: Although the use of Gly is beneficial as indicated by previous reports, we concluded that it does not meet the requirement for proper fixation, at least for the tested cell types and proteins. However, PFA alone with no addition of TX displayed a significant cytoplasmic loss by generating membrane blebs during fixation.

Spatiotemporal expression and regulation of FoxO1 in mouse uterus during peri-implantation period.

Recent studies indicate that FoxO1 has roles in female reproductive system, especially in maternal endometrium. Although various cellular aspects and molecular pathways have been identified, the exact molecular characteristics of embryo implantation are still not completely understood. In this study, we aimed to investigate uterine expression and regulation of FoxO1 during peri-implantation period in mice. Experimental mouse models including, normal pregnancy, pseudopregnancy, artificial decidualization, and delayed implantation and activation were performed. Our results showed that FoxO1 expression was spatiotemporal in mouse endometrial tissue throughout peri-implantation period and its expression was significantly upregulated in luminal and glandular epithelium at the time of implantation. Moreover, on day 5 morning (09:00 AM) of pregnancy, expression of FoxO1 was cytoplasmic in endometrial luminal epithelial cells where embryo homing takes place. With progressing time on day 5 evening (19:00 PM) of pregnancy FoxO1 expression was nuclear in luminal epithelium at implantation site. Pseudopregnancy and artificial decidualization models indicated that FoxO1 expression was regulated by pregnancy hormones. Delayed implantation and activation model indicated that FoxO1 expression at the time of implantation is dependent upon activation status of blastocyst due to E2 induction and uterine sensitivity to implantation. In conclusion, our findings highlight a perspective for FoxO1 expression and regulation in mouse uterus during peri-implantation period indicating that its expression is regulated by implanting embryo and pregnancy hormones.

Random start controlled ovarian hyperstimulation for fertility preservation during incidental pregnancy: a case report of blastocyst vitrification from in vitro matured oocytes.

Here, we present a diffuse large B cell lymphoma patient admitted for fertility preservation before cancer therapy and whose pregnancy was recognized incidentally just after the start of random start controlled ovarian stimulation (RSCOH) during the stimulation cycle. Despite an optimal homogenous growth of follicle cohort, majority of the retrieved oocytes were immature after GnRHa trigger. Possible effects of extremely high serum progesterone and/or ß-hCG levels on oocyte in vivo maturation are discussed with the surprising high rate of in vitro maturation and subsequent good embryo development. It seems that in case of need for pregnancy termination as a result of an urgent cancer therapy, RSCOH can be started and patients may benefit from overnight in vitro maturation of oocytes.

ADAMTS1 and ADAMTS5 metalloproteases produced by Sertoli cells: a potential diagnostic marker in azoospermia.

In this study, our aim was to detect protein levels of A Disintegrin and Metalloproteinase with Thrombospondin Motifs 1 and 5 (ADAMTS1 and ADAMTS5) proteases and to examine the effect of in vitro FSH supplementation on protease production in cultured Sertoli cells. The expression of metalloproteases, ADAMTS1, and ADAMTS5 were investigated in Sertoli cell cultures as well as in ejaculate of azoospermic men which then were compared with ejaculates of the fertile control group. A total of 15 azoospermic men, diagnosed as obstructive (OA, n = 5) and nonobstructive (NOA, n = 10) azoospermia were included in the study. ADAMTS1, ADAMTS5 and FSH receptors (FSHR) were found to be expressed 2.56, 2.10, and 2.66-fold less in Sertoli cells of NOA patients, than those of OA (p < 0.05). After rFSH was added onto Sertoli cell cultures of NOA patients, their expression did not increase significantly and did not reach to levels of control group. Evaluation of ejaculates revealed that the expression of ADAMTS1 and ADAMTS5 were insignificantly 1.03 and 1.1-fold higher in OA group (p > 0.05), respectively; however, in the NOA group, their expression were 1.70 and 1.96-fold lower, respectively, when compared with the fertile control group (p < 0.05) which was statistically significant. As a conclusion, the present study has revealed that insufficiency of ADAMTS1 and ADAMTS5 expression in Sertoli cells may have an important role in the etiology of male infertility. As expected due to low FSHR expression, rFSH response is impaired in NOA patients with relatively low ADAMTS expression response; therefore, such patients might hardly benefit from rFSH treatment. Further studies with larger cohorts may reveal ADAMTSs' potential use as a predictive marker for positive sperm retrieval in azoospermic patients who are scheduled to undergo testicular sperm extraction. Abbreviations: ADAM: A Disintegrin and Metalloproteinase; ADAMTS1 and ADAMTS5: A Disintegrin and Metalloproteinase with 10 Thrombospondin Motifs 1 and 5; ADAMTS: A Disintegrin and Metalloproteinase with Thrombospondin; ABP: androgen binding protein; CAMs: cell adhesion molecules; ECM: extracellular matrix; FSH: follicle stimulating hormone; FSHR: FSH receptors; HRP: horseradish peroxidase; MMP: matrix metalloproteinases; MP: metalloproteinases; NOA: nonobstructive azoospermia; OA: obstructive azoospermia; TIMP-1: tissue inhibitor of metalloproteinase-1.

Expression of inhibitor proteins that control primordial follicle reserve decreases in cryopreserved ovaries after autotransplantation.

PURPOSE: Even with 86 live births reported globally so far, the mechanism of primordial follicle loss following autotransplantation of the frozen-thawed ovarian tissue needs further evaluation. Pten, Tsc1, p27, and Amh are the inhibitor proteins that play crucial roles in suppressing the transition from the primordial follicle to primary state, maintaining the primordial follicle reserve. In this study, we aimed to evaluate whether the expression patterns of these proteins change and it may be related to the global primordial follicle loss after autotransplantation of the frozen-thawed ovarian tissue. METHODS: Four groups were established in rats: fresh-control, frozen/thawed, fresh-transplanted, and frozen/thawed and transplanted. After slow freezing and thawing process, two ovarian pieces were transplanted into the back muscle of the same rat. After 2 weeks, grafts were harvested, fixed, and embedded into the paraffin block. Normal and atretic primordial/growing follicle count was performed in all groups. Ovarian tissues were evaluated for the dynamic expressions of the Pten, Tsc1, p27, and Amh proteins using immunohistochemistry, and H-score analyses were done. RESULTS: Ovarian tissue cryopreservation does not change the expression patterns of inhibitory proteins that control ovarian reserve. Both in fresh and frozen/thawed autotransplanted groups, the expression of inhibitory proteins and Amh decreased significantly in primordial follicles and in growing follicles, respectively. In control group and in frozen/thawed group, primordial follicle counts were similar but decreased by almost half in both fresh-transplanted and frozen/thawed and transplanted groups. CONCLUSIONS: One of the causes of primordial follicle loss after transplantation of ovarian graft may be decreased expression of the inhibitory proteins that guard the ovarian reserve and transplantation itself seems to be the major cause for disruption of inhibitory molecular signaling. Our findings highlight important molecular aspects for future clinical applications for fertility preservation in humans.

Live birth after Laser Assisted Viability Assessment (LAVA) to detect pentoxifylline resistant ejaculated immotile spermatozoa during ICSI in a couple with male Kartagener's syndrome.

Primary ciliary dyskinesia (PCD) is a rare, autosomal recessive disease with abnormalities in the structure of cilia, causing impairment of muco-ciliary clearance with respiratory tract infections, heterotaxia and abnormal sperm motility with male infertility. Here, with a comprehensive literature review, we report a couple with an infertility history of 9 years and three unsuccessful IVF treatments, where male partner has Kartagener's Syndrome, a subtype of PCD, displaying recurrent respiratory infections, dextrocardia and total asthenozoospermia. His diagnosis was verified with transmission electron microscopy and genetic mutation screening, revealing total absence of dynein arms in sperm tails and homozygous mutation in the ZMYND10, heterozygous mutations in the ARMC4 and DNAH5 genes. Laser assisted viability assay (LAVA) was performed by shooting the sperm tails during sperm retrieval for microinjection, following detection of pentoxifylline resistant immotile sperm. Live births of healthy triplets, one boy and two monozygotic girls, was achieved after double blastocyst transfer.