Fertility preservation in cancer patients: in vivo and in vitro options

Oocyte, embryo and ovarian tissue cryopreservation are being increasingly proposed for fertility preservation among cancer patients undergoing therapy to enable them to have babies after the cancer is cured. Embryo cryopreservation is not appropriate for single girls without any spermpartner. It is impossible in cases requiring immediate cancer cure because oocyte retrieval is an extended procedure. Thus ovarian tissue cryopreservation has been suggested for fertility preservation especially in cancer patients. The main goal of ovarian cryopreservation is re-implanting the tissue into the body to restore fertility and the hormonal cycle. Different cryopreservation protocols have been examined and established for vitrification of biological samples. We have used Cryopin to plunge ovarian tissue into the liquid nitrogen and promising results have been observed. The possibility of recurrence of malignancy in the reimplanted tissue could be a problem. Xenografting-implantation of the preserved tissue in another species-also has its drawbacks such as molecular signaling from the recipient. In vitro follicle culturing is a safer method to obtain mature oocytes for fertilization and the various studies that have been carried out in this area are reviewed in this paper

Effect of prolonged culture of chromosomally abnormal human embryos on the rate of diploid cells

Background: A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies

Materials and Methods: In this cohort study, we used fluorescence in situ hybridization [FISH] to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis [PGD] on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization

Results: Of the 30 analyzed blastocysts, mosaicism was observed in 26[86.6%], while 2[6.7%] were diploid, and 2[6.7%] were triploid. Of those with mosaicism, 23[88.5%] were determined to be diploid-aneuploid and 3[11.5%] were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 [P<0.05]; however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality

Conclusion: Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells

introduction to the royan human ovarian tissue bank

From December 2000 until 2010, the researchers at Royan Institute conducted a wide range of investigations on ovarian tissue cryopreservation with the intent to provide fertility preservation to cancer patients that were considered to be candidates for these services. In 2010, Royan Institute established the Royan Human Ovarian Tissue Bank as a subgroup of the Embryology Department. Since its inception, approximately 180 patients between the ages of 747 years have undergone consultations. Ovarian samples were cryopreserved from 47 patients [age: 7-35 years] diagnosed with cervical adenocarcinoma [n=9]; breast carcinoma [n=7], Ewing's sarcoma [n=7], opposite side ovarian tumor [n=7], endometrial adenocarcinoma [n=4], malignant colon tumors [n=3], as well as Hodgkin's lymphoma, major thalassemia and acute lymphoblastic leukemia [n=1-2 patients for each disease]. Additionally, two patients requested ovarian tissue transplantation after completion of their treatments

Follicle development of xenotransplanted sheep ovarian tissue into male and female immunodeficient rats

This study aimed to assess follicle survival after xenotransplantation of sheep ovarian tissue into male and female immunodeficient rats. We evaluated the effects of gonadotropin treatment on follicular development in the transplanted tissue. In this experimental study, sheep ovarian cortical strips were transplanted into the neck back muscles of 8 male and 8 female immunodeficient, castrated rats. Fourteen days after surgery, each rat was treated with human menopausal gonadotropin [hMG] for 9 weeks. One day after the last injection, ovarian tissues were removed and fixed for histology assessment. Histology analyses were performed before and after grafting. Estradiol [E[2]] levels were measured before and after gonadectomy, and at the end of the experiment. The control group consisted of 7 male and 7 female non-castrated/non-grafted rats and the sham group comprised 7 male and 7 female castrated/ non-grafted rats for comparison of serum E2 concentrations. The percentage of primordial follicles decreased after transplantation in male [25.97%] and female [24.14%] rats compared to the control group [ovarian tissue non-grafted; 37.51%]. Preantral follicles increased in the male [19.5%] and female [19.49%] transplanted rats compared to the control group [11.4%]. Differences in antral follicles between male [0.06 +/- 0.0%] and female [0.06 +/- 0.0%] rats were not noticeable compared to control [1.25 +/- 0.0%] rats. We observed a significantly higher percent of mean E[2] secretion in grafted males compared to grafted females [P<0.05]. Despite significant differences in E[2] secretion between xenografted male and female rats, we observed no statistical differences in terms of follicular development

Relationship between seminal melatonin with sperm parameters, DNA Fragmentation and nuclear maturity in Intra-Cytoplasmic sperm injection candidates

Objective: Melatonin, the chief secretory product of the pineal gland, regulates dynamic physiological adaptations that occur in seasonally breeding mammals as a response to changes in daylight hours. Because of the presence of melatonin in semen and the membrane melatonin receptor in spermatozoa, the impact of melatonin on the regulation of male infertility is still questionable. The aim of this study was to determine the effects of endogenous melatonin on human semen parameters [sperm concentration, motility and normal morphology], DNA fragmentation [DF] and nuclear maturity

Materials and Methods: In this clinical prospective study, semen samples from 75 infertile men were routinely analyzed and assessed for melatonin and total antioxidant capacity [TAC] levels using the enzyme-linked immunosorbent assay [ELISA] and colorimetric assay kits, respectively. DF was examined by the sperm chromatin dispersion [SCD] test. Acidic aniline blue staining was used to detect chromatin defects in the sperm nuclei

Results: There was no significant correlation between seminal plasma melatonin and TAC with sperm parameters and nuclear maturity. However, we observed a positive significant correlation between DF and melatonin level [r=0.273, P<0.05]

Conclusion: Melatonin in seminal plasma is positively correlated with damaged sperm DNA of infertile patients. The mechanism of this phenomenon needs further study

[ review on the activity of hormones and growth factors after ovarian tissue transplantation]

The majority of cancer treatments are invasive. Gonadal injuries cause reductions in fertility which results in lack of hope for conception in cancer patients and frustration for their partners. Fortunately, current advancements in cryopreservation and transplantation sciences regarding fertility preservation lead to cryostorage of gonads and preservation prior to the onset of chemo- and radiotherapy treatments. Accordingly in women, the main goal of ovarian cryopreservation is establishment of fertility and hormonal cycle restoration after auto-transplantation. Although the history of ovarian transplantation dates back to the 19[th] century, there are reports of live human births following ovarian tissue cryopreservation and transplantation since the past 100 years. Despite this success and additional research in the field of ovarian cryopreservation and transplantation, numerous questions remain unanswered. Among these questions, growth factors and hormonal changes because of their effects on follicular function appear to be more important during ovarian tissue transplantation. This review attempts to address hormones and growth factor functions with the specifics of ovarian cryopreservation and auto-transplantation

In vitro maturation, fertilization and embryo culture of oocytes obtained from vitrified auto-transplanted mouse ovary

The purpose of this study was to investigate the in vitro survival and developmental potential of oocytes obtained from vitrified mouse ovaries transplanted to a heterotopic site. In this experimental study, two-week-old mice were unilaterally ovariectomized after anesthesia. The ovaries were vitrified by cryotop. After two weeks, the ovaries were thawed and autotransplanted to the gluteus muscle tissue. Three weeks later the mice were killed, after which we removed and dissected the transplanted and opposite right ovaries. Cumulus oocyte complexes [COCs] and denuded oocytes were evaluated for in vitro maturation [IVM], in vitro fertilization [IVF] and in vitro development [IVD]. The control group consisted of sevenweek- old age-matched mice ovaries. All vitrified-transplanted [Vit-trans] ovaries contained some oocytes that survived. Following IVM, IVF and IVD, there were 41.7% out of 12 cultured zygotes that reached the 8-cell stage. Our experiment supports the progressive role of long-term graft survival after wholeovarian cryopreservation by vitrification and subsequent heterotopic transplantation. It is possible to recover viable follicles and oocytes that have the ability to develop in vitro

effects of ISM1 medium on embryo quality and outcomes of IVF/ICSI cycles

The aim of this study is to investigate the effect of ISM1 culture medium on embryo development, quality and outcomes of in vitro fertilization/intracytoplasmic sperm injection [IVF/ICSI] cycles. This study compares culture medium commonly used in the laboratory setting for oocyte recovery and embryo development with a medium from MediCult. We have assessed the effects of these media on embryo development and newborn characteristics. In this prospective randomized study, fertilized oocytes from patients were randomly assigned to culture in ISM1 [MediCult, cycles: n=293] or routine lab culture medium [G-1[TM]v5; Vitrolife, cycles: n=290] according to the daily media schedule for oocyte retrieval. IVF or ICSI and embryo transfer were performed with either MediCult media or routine lab media. Embryo quality on days 2/3, cleavage, pregnancy and implantation rates, baby take home rate [BTHR], in addition to the weight and length of newborns were compared between groups. There were similar cleavage rates for ISM1 [86%] vs. G-1[TM] v5 [88%]. We observed a significantly higher percentage of excellent embryos in ISM1 [42.7%] compared to G-1[TM] v5 [39%, p<0.05]. Babies born after culture in ISM1 had both higher birth weight [3.03 kg] and length [48.8 cm] compared to G-1[TM] v5 babies that had a birth weight of 2.66 kg and a length of 46.0 cm [p<0.001 for both]. This study suggests that ISM1 is a more effective culture medium in generating higher quality embryos, which may be reflected in the characteristics of babies at birth

Sucrose effect on follicular survival rate and apoptosis incidence in rat ovarian tissue after vitrification

the aim of this study was to evaluate the effect of sucrose on follicular survival rate and the incidence of apoptosis in rat ovarian tissue following vitrification. Ovaries of approximately 5-week-old female Wistar rats were divided randomly into three groups: control [non-vitrified], V[I] [Ethylene Glycol + Dimethyl Sulfoxide] and V[II] [EG + DMSO + 0.25 mol/lit sucrose]. Vitrified-warmed samples were incubated for approximately 30 minutes and fixed in Bouin's fixative. The samples were serially sectioned and stained either with H and E or immunohistochemistry kit of anti-active and a pro-caspase-3 kit. Data analysis showed that the rate of growing follicles that survived, with the exception of primordial follicles, was comparable between the vitrified-warmed and control samples. Morphologically healthy primordial follicles showed significant reductions in all vitrification groups compared to the control, however this rate was not significant between the vitrification groups. In comparison with healthy follicles, there were significantly more dead follicles in the vitrification groups than the control group. In addition the apoptotic follicles increased significantly after vitrification, with the exception of the antral follicles. Although the number of apoptotic follicles was similar between both vitrification groups, however there were significantly more pre-antral apoptotic follicles in the VII group compared to the VI and control groups. According to these results, the presence or absence of sucrose has no significant effect on the preservation of primordial and primary follicles which are important for transplantation

[Temperature reduction process - a way for fertility preservation in cancer patients]

More than the half of cancer patients undergo cancer treatments of chemotherapy and/or radiotherapy. Unfortunately treatment with invasive methods occasionally lead to severe side effects. Patients who undergo chemotherapy can be affected by premature ovarian failure, an important cause of infertility. Ovarian tissue cryopreservation is suggested as the only way for preservation of sex cells and fertility preservation in cases of prepubertal girls and women with sterility attributed to chemotherapy, radiotherapy, genetic disorders or specific diseases

epididymal sperm viability, motility and DNA integrity in dead mice maintained at 4-6[degree sign]C

When male animals die, spermatozoa within the body of animal will be degenerated. Because of unique chromatin structure of sperm, maybe this degeneration is different from other cells. However there is not any research which considered directly the integrity of sperm DNA by keeping the cadaver in refrigerator. The aim of this study was to assess viability, total motility and DNA integrity of sperm cells after death. In this experimental study, 24 male Swiss white mice were killed by cervical dislocation and then kept in refrigerator [4-6[degree sign] C] for up to 12 days. On the 0 [immediately after death as control group], 1[st], 2[nd], 3[rd], 5[th], 7[th], 10[th] and the 12[th] days after death cauda epididymides were removed and squeezed in Ham's F10 medium. The proportion of viable, motile and double stranded DNA spermatozoa was examined. Viability and DNA integrity of sperm cells were examined consecutively by eosin nigrosin and acridine orange stainings. The data obtained from this study showed that viability and total motility of sperm cells were significantly decreased during 12 days after death [p<0.001]. In contrast with viability and motility, DNA integrity was without significant changes [even 12 days after death]. This study suggests that integrity of sperm DNA would not change even after 12 days after death if the cadaver kept in refrigerator

[ effects of Taurine antioxidant on standard parameters, DNA fragmentation, Protamine deficiency and oxidative stress on freezing human semen]

Evaluation of the effects of different doses of antioxidant Taurine on oxidative stress and human sperm parameters following cryopreservation. The semen of 20 fertile men were divided to 5 aliquot, one part considered as a fresh after analysis of standard semen parameters [Motility, Abnormal Morphology, Viability] and Protamine deficiency, DNA damage and measurement of ROS and RNS. The other part was loaded on to a 80% and 40% Allgrad gradient and centrifuged. The pellet was washed and divided into 4 separate fractions for control [non-frozen] and cryopreservation groups in absence or presence of 0.25 and 50 mM Taurine. The frozen specimen were thawed and then examined. Sperm Motility evaluated using a CASA software. The Viability of spermatozoa was assessed by the Trypan-Blue stain method. Levels of ROS determined by spectroflorometry assay using DCFH-DA. DNA fragmentation examined by SCD test and Protamine deficiency examined by CMA3+ staining. At the end results were analyzed using ANOVA test. Cryopreservation procedure increased the amount of ROS and adding 25 mM of Taurine improved post-thaw motility, progressive motility and sperm protamine deficiency. However, different doses of Taurine [25 and 50 mM] had no significant effects in total abnormalities, viability, DNA fragmentation and ROS reduction. Antioxidant Taurine has no significant effects on ROS production following human sperm cryopreservation. But with dose of 25mM could improve the quality of spermatozoa due to the assessment of motility and protamine deficiency

[Quantitative expression of BAG1, BAX and BCL-2 genes in human embryos with different fragmentation grades derived from ART]

The purpose of this study was to evaluate the quantitative expressions of BAG1, BAX and BCL-2 in human embryos with different fragmentation grades as derived from assisted reproduction technology [ART]. Fragmented and normal human 8-cell embryos were scored according to the degree of fragmentation with an inverted microscope and divided into four grades [grade I: no or minimal fragmentation [<5%], grade II: embryos with <25% fragmentation, grade III: embryos with >25% fragmentation and grade IV: apoptotic induced embryos with actinomycin D]. In this study, TUNEL labeling was initially used to detect apoptosis, and then revers transcription polymerase chain reaction [RT-PCR] and quantitative PCR were used to define the quantitative expressions of experimental genes in human embryos with different fragmentation grades. The results of TUNEL labeling showed that embryos with higher fragmentation had a high number of apoptotic bodies. The results of RT-PCR and q-PCR analyses showed a significantly decreased amount of BAGI transcript expression from group I to group IV. The highest expression of BAX gene was observed in group II, however, the transcript of BCL-2 gene was not observed in any of the experimental groups. The effect of actinomycin D on transcript expression amounts of experimental genes in apoptotic induced embryos [group IV] compared to control embryos [group I] showed a significant decrease. mRNA expression of BAG1 gene can be used as a good marker to detect apoptosis in human embryos. However, the transcript of BCL-2 gene does not play a role in the detection of apoptosis in human embryos at the 8-cell stage

[ role of ultraweb nano-fibrillar substrates in the differentiation of in vitro mouse bone marrow mesenchymal stem cell-derived hepatocyte-like cells]

To study the impact of ultraweb nano-fibrillar substrates on in vitro differentiation of mouse bone marrow-derived mesenchymal stem cells into hepatocyte-like cells. For a duration of up to 36 days, mouse bone marrow mesenchymal stem cells [MSCs] were cultured on an artificial basement membrane containing ultraweb nanofiber [nano+] and plates [nano] coated with Matrigel and used growth and differentiating factors for hepatogenic differentiation. Then, to study the impact of nanofibers on hepatocyte differentiation, mRNA expressions for liver-specific genes [CK7, AFP, FOXa2, ALB, CYP1a1, HNF4a] were analyzed using real-time PCR, and ultrastructures of differentiated cells were evaluated using scanning and transmission electron microscope. Both experimental groups expressed mRNA of liver-specific genes in a time dependent manner. However, mRNA expression trends of liver-specific genes, especially those of FOXa2 and HNF4a, were better in the nano+ culture as compared to the nanoculture [p<0.05]. Furthermore, the ultrastructures of differentiated cells in the 36 day-old nano+ culture were more mature as compared to those of the nanogroup. The results suggest that topographical properties of the extracellular matrices produced by ultraweb nanofibers are effective for the in vitro reproduction of more differentiated MSC-derived hepatocyte-like cells

correlation between the endometrial integrins and osteopontin expression with pinopodes development in ovariectomized mice in response to exogenous steroids hormones

The ovariectomized animals are good models to evaluate the effect of different steroid hormone treatments on implantation events and the pattern of integrin expression. Therefore, this study was performed to compare the expression of integrins and osteopontin [OPN] in correlation with pinopode development in ovariectomized mice endometrium which was subjected to steroid hormones. Ovariectomized mice were subjected to estrogen, progesterone and estrogen-progesterone hormones. Their uterine horns were evaluated for integrin expression by immunohistochemistry and real-time RT-PCR and for pinopode development by transmission and scanning electron microscopic studies. No immunostaining for integrin and OPN molecules were detected in the endometrium of non-ovariectomized mice except in metestrus phase. The alpha4 and beta 1 integrin genes were expressed in all phases of estrous cycle. In ovariectomized mice, no reaction was detected in the endometrium of control, sham and estrogen-treated groups, but in both progesterone-treated groups, all examined genes were expressed. There was not any correlation between pinopodes and integrin expression in ovariectomized mice. The progesterone is more effective on endometrial integrin expression than estrogen and differences in the expression pattern of integrins reflect their important and different roles in embryo implantation. The pinopodes may have minor effect in mice implantation or have some delay in their expressions in ovariectomized mice which were subjected to exogenous hormones

Transplantation and homing of mouse embryonic stem cells treated with erythropoietin in spleen and liver of irradiated mice

The present study was designed to evaluate the homing potential of mouse embryonic stem cells [ESC] treated with erythropoietin [EPO] in hematopoietic organs such as spleen and liver after transplantation using morphological and immuno-histochemical techniques. Day-four embryoid body [EB]-derived cells were dissociated and re-plated in medium in the presence and absence of EPO for three days. The EPO- and untreated differentiated cells were labeled with 5-bromo-2 deoxyuridine [BrdU] before transplantation and analyzed using flow cytometry and reverse transcription-PCR methods. BrdU-labeled cells were injected via the tail vein into irradiated adult mice in both groups. The spleen colony-forming unit assay [CFU-S] was performed 12 days after transplantation. Immuno-histochemistry was also carried out to trace transplanted cells. The percentage of CD34 positive cells was 5.51 +/- 1.06% in the EPO-treated group and 1.63 +/- 0.225% in untreated group. The RT-PCR analysis showed that the EPO-treated cells expressed epsilon globin, beta H1 globin, RUNX1 and EPO receptor genes, but the beta-major globin gene was not expressed. The number of colonies formed in the spleens of treated group [17.33 +/- 4.726] was significantly different from the control group [6 +/- 1]. The population of BrdU positive cells in spleen of EPO-treated cell-transplanted group was higher than that of the control group. Also, BrdU positive cells were observed in the central vein of the liver sections of EPO-treated and control groups but were not observed in the liver parenchyma. There were not BrdU positive cells in the spleen and liver sections of the sham group. Our results confirm that ESC have the ability to home and form colonies in spleen after transplantation and EPO-treated EB-derived cells caused an increase in the number of colonies in spleen after CFU-S

Endometrial receptivity to implantation in humans: biochemical and molecular aspects

The success rate of several advanced basic and clinical techniques in the field of mammalian biotechnology, including cloning, pre-implantation genetic diagnosis, and assisted reproductive techniques [ART] depends mainly on the success rate of pregnancy following in vitro fertilization-embryo transfer [IVF-ET]. The techniques used in ART have advanced considerably since the first in vitro fertilization birth in 1978. However, despite these advances, pregnancy rates are still relatively low and have not increased significantly in the last decade. Based on the facts that embryo implantation is considered as the last barrier in ART and that inadequate endometrial receptivity is responsible for approximately two-thirds of implantation failures, intensive research work has been performed to understand the physiology, regulation, and the clinical assessments of the endometrial receptivity to improve the success rate of IVF-ET. This and the ongoing reviews tend to cover the different aspects of the endometrial receptivity mainly in human model. The present part of this series primarily concerns with biochemical and molecular events in the endometrium coordinated within its receptivity period termed as the window of implantation. Successive sections will deal with its ultrastructural changes, biomarkers, clinical assessments and regulators of endometrium within the window of implantation

Comparison of embryo development between intracytoplasmic and piezo-assisted sperm injection after treating mouse sperms by Ca[2+] ionophore

The purpose of this study was to evaluate the efficiency of intracytoplasmic sperm injection [ICSI] and Piezo-assisted sperm injection after pretreatment with calcium ionophore [CaI] on the mouse embryo development. In this study, the conventional ICSI and Piezo-ICSI procedures were used. The efficacy of the methods was examined after mouse matured oocytes were fertilized with or without CaItreated sperms. Piezo-ICSI demonstrated significantly more favorable results, with a fertilization rate of 64% [conventional ICSI: 42%, P<0.001] and a cleavage rate of 73% [conventional ICSI: 58%, P<0.05]. When the Piezo-ICSI procedure was performed with CaI-pretreated sperms, the cleavage rate significantly increased [92% vs. 73%, P<0.05]. However, the fertilization rate did not change significantly [64% vs. 56%]. The Piezo-ICSI accompanies with CaI-treated sperms is more efficient than the conventional ICSI method for fertilizing and thus obtaining more mouse embryos

effects of different concentrations of leukemia inhibitory factor on the development of isolated preantral follicles from fresh and vitrified mouse ovaries

Leukemia inhibitory factor [LIF] is a pleiotropic cytokine of interleukin-6 family with a remarkable range of biological actions such as proliferative effects on the granulosa and theca cells. The aim of this study was to evaluate the effects of LIF on the growth and maturation of mouse fresh and vitrified preantral follicles, an in vitro model was developed. The ovaries of 14-day-old mice were vitrified in a mixture of ethylene glycol, ficoll 70 and sucrose in PB1 for 5 min. The preantral follicles were mechanically isolated from vitrified-warmed and non-vitrified ovaries. They were cultured in alpha-minimum essential medium supplemented with 5% fetal bovine serum, 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin and selenium, 20 ng/ml murine recombinant epidermal growth factor and different concentrations of LIF [25, 50, 100 ng/ml] for 12 days. On day 12, ovulation was induced using 1.5 mIU/ml human chorionic gonadotropin. In this study, the follicle diameter, survival rate and maturation rate were assessed. The mean diameter of fresh and vitrified preantral follicles cultured in 50 ng/ml concentration of murine recombinant LIF was significantly higher than that of other concentrations in each group on day 2 [229.42 +/- 30.40, 222.55 +/- 33.4] [P<.001] and on day 4 [340.45 +/- 61.05, 299.50 +/- 65.55], respectively [P<0.01]. The survival rates of follicle in fresh and vitrified groups were 80.56% and 77.78, respectively. There was no significant difference between control and treated groups. The percentage of follicles which released metaphase II [MII] oocyte in fresh groups in the presence of 0, 25, 50, 100 ng/ml of LIF was 16%, 14.28%, 40% and 21.05% [P<0.01] and so in vitrified groups were 11.76%, 14.28%, 28.57% and 13.38%, respectively [P<0.05]. There were significant differences between 50 ng/ml LIF-treated groups with other concentrations in each group. Therefore, in vitro growth and maturation of mouse follicles were improved in the presence of 50 ng/ml LIF