Background: Semen cryopreservation is a useful tool for preserving fertility for men involved in ART program who have been diagnosed with cancer and will undergo chemotherapy, radiotherapy or testicular surgery. Objective: The objective of this study to evaluate the effect of honey bee supplemented to cryoprotectant medium on post-thaw sperm DNA integrity ,morphology and round cells. Subjects ,Materials and Methods: Thirty semen samples were collected from 30 infertile patients. After assessment of semen analysis, semen samples were divided into 3 aliquots (0.7ml for each sample) and mixed with 1 ml of cryopreservation solution (G1,control) alone, enriched with 5% honey bee(G2) with 10% honey bee (G3) for cryopreservation. Current study used the acridine orange test to investigate the effect of honey bee in two concentration (5%, 10%) on the integrity of sperm chromatin structure pre-and post cryopreservation. Cryopreservation was done at -196 Co in liquid nitrogen and thawing was performed after six months. Direct swim up technique was used for in vitro sperm preparation post-thawing, sperm parameters were assessed and data were statistically analyzed pre-and post-thawing. Results: The results show there was a significant(P<0.05) increase in the percentage of morphologically normal sperm for all groups post-thawing, particularly for G3 group that was significantly (P<0.05) increased as compared to G1 and G2 groups, In contrast, non-significant differences (P>0.05) were observed between G1 and G2 groups. Sperm DNA fragmentation percentage (%) was significantly(P<0.05) increased in G1 post thawing without any additives as compared to pre-cryopreservation , where as in G2 and G3 sperm DNA fragmentation were increased but not significantly as post-cryopreservation groups. Also the results show that there were no significant differences between G2 and G3 whereas G3 gives better result as compared to G1 with significant differences. Round cells counts for all groups of post cryopreservation were significantly(P>0.05) decreased as compared to pre-cryopreservation. Non significant (P>0.05) differences in the round cells counts among all the groups of post -thawing in spite of G3 group has the lowest mean. Conclusions: From these results we concluded that supplementation of 10% of honey bee to the freezing cryoprotectant medium gives best protection to the morphology and DNA integrity of human sperm.
Background: Chemotherapy and radiotherapy can destroy or severely reduce spermatogenesis and thereby jeopardize fertility in the long term. There is still no medical treatment that guarantees fertility preservation after chemo- and radiotherapy. Now with recent improvement of assisted reproductive technologies (ART) and possibility of using testicular spermatozoa or epididymal spermatozoa at in vitro fertilization (IVF) by intracytoplasmic sperm injection (ICSI), cryopreservation of testicular tissue is an option in fertility preservation for males who will lose spermatogenic cells as a result of chemoand radiotherapy and for males with azoospermia. Objectives: This study is an attempt to evaluate oxidative DNA damage in the mice testicular tissue after cryopreservation/thawing cycle when using different types of cryoprotectants as well as to investigate the changes that occur in mice testicular tissue after cryopreservation/thawing cycle as a model for human being. Methods: Fifty mature fertile male mice between 8- 12 weeks old were used in the current study. For each mouse, one testis was evaluated histologically and immunohistochemically (using 8-Hydroxy 2'-Deoxyguanosine as marker of oxidative DNA damage) without cryopreservation (control group). The other testis was evaluated histologically and immunohistochemically (using 8-Hydroxy 2'-Deoxyguanosine as marker of oxidative DNA damage) after six weeks of cryopreservation using different types of cryoprotectants (cryopreserved group). Results: The microscopically observation of slides obtained from cryopreserved testis differs according to the type of cryoprotectant (glycerol, 1,2 propanediol and dimethylsulfoxide), in the dimethylsulfoxide group, tissue sections showed no major differences versus control group, but in the histological sections obtained from tissue cryopreserved by glycerol as cryoprotectant showed moderate morphological and structural changes as compared with the control group. While, the tissue samples subjected to 1,2 propanediol as cryoprotectant displayed the severest morphological and structural changes as compared with the control group. Immunohistochemical results of the present study showed a highly significant (P<0.001) increase in oxidative DNA damage in the cryopreserved testis (46.78%, 63.45% and 32.59% represent cryoprotectant glycerol, 1, 2 propanediol and dimethylsulfoxide, respectively) compared with control group (19.27%) after six weeks of cryopreservation. Conclusion: From the results of the current study, it was concluded that there was alteration in testis histology after cryopreservation/thawing cycle and increase the level of oxidative DNA damage after cryopreservation/thawing cycle. As well as dimethylsulfoxide cryoprotectant provide good protection for testis histology and DNA.
Background: All men have the natural desire to have their own biological children. Cryopreservation of male gametes is an important aspect of human fertility preservation. With the advancement in assisted reproductive technology, indication for sperm cryopreservation is expanding. Objectives: To evaluate the effect of cryopreservation by using Sperm Freeze TM kit on some sperm parameters for infertile patients. Materials and methods: Ninety five semen samples were taken from male patients. The seminal fluid analysis was performed according to the standard criteria of WHO (1999). The patients were divided into four groups depending on their sperm concentration and motility: Normozoospermic, oligozoospermic, asthenozoospermic and oligoasthenozoospermic patients. Cryopreservation in liquid nitrogen was done by using Sperm Freeze TM kit for the samples and thawing was performed after three months. Results: In general, the post-thawing study revealed a considerable highly significant (P <0.001) decrease in the sperm parameters which include the means of sperm concentration, sperm motility, progressive motility and normal sperm morphology as compared to that of pre-cryopreservation. Whereas the percentage of immotile sperm was highly significant (p<0.05) increase as compared to pre-cryopreservation in all groups except for oligozoospermic patients in which there was a significant (p<0.05) increase. Conclusions: After semen cryopreservation, all parameters were significantly decreased.
Background: The semen cryopreservation has an important role in the male fertility preservation. Certainly, patients who are prone to become infertile due to surgical or medical treatments such as chemo- or radiotherapy for cancer treatment. In fact, semen cryostorage seems to be the only proven method that may offer these couples a chance of having children in the future. Objectives: This study was aimed to investigate the effects of cryopreservation protocols on human sperm parameters. Methods: Sixty semen samples with range of age (19-35) years were included in this study. Sperm parameters, were assessed pre- or post-cryopreservation. Cryopreservation protocols were done using SMART medium with either 15% dimethyl sulfoxide(DMSO) or 15% glycerol alone (as control group) or supplemented with either 0.25M or 0.5M of sucrose for treated groups. Crude data were statistically analyzed. Results: Generally, the results of the present study showed that the sperm parameters were significantly deteriorated post-thawing (P<0.05) as compared to precryopreservation. Both treated groups (G2 and G3) showed best results as compared to G1 (control groups). In contrast, treated group (G2) with 0.25 M of sucrose appeared better results than the as G3 group that treated with 0.5M of sucrose. Conclusion: Sperm parameters including concentration, motility, and morphology were deteriorated after cryopreservation. Using 0.25M sucrose reduced the impact of cryopreservation.
Background: Many studies conducted on the pentoxifylline (PX) and Glycyrrhiza glabra (G.glabra) as motility stimulants showed positive effect on sperm preparation in vitro improving the progressive forward movement. However, it was not known the impact of these stimulants on the nature of genetic material, especially after cryopreservation. Objective: The experiment was designed to find out any harmful effect of a medium containing a mixture of PX and aqueous extract of G.glabra on the sperm DNA before and after cryopreservation. Materials and Methods: Fifty mature fertile male mice 8-12 weeks old were used. Epididymal sperms were obtained from caudal region and prepared in vitro by direct activation technique using four media namely; PX alone, G.glabra alone, a mixture of PX with G.glabra and Ham's F-12 (as control medium). Certain sperm function characteristics were examined as well as evaluation of DNA integrity by acridine orange test and comet assay before and after cryopreservation. Results: The results showed a highly significant (P<0.001) improvement in the sperm forward movement, with a highly significant (P<0.001) decrease in DNA abnormality using acridine orange test and comet assay, following in vitro activation by the four media compared to before activation. Following in vitro activation and cryopreservation, the results of epididymal sperm showed a highly significant (P<0.001) decrease in DNA abnormality by using PX medium alone, G.glabra medium alone and the mixture of PX and G.glabra compared to control medium and the results before cryopreservation. Conclusion: According to the results of present study, it has been found no toxic effect of medium containing PX and/or G.glabra on the epididymal sperms DNA after activation in vitro and cryopreservation using acridine orange test and comet assay. The best result obtained when using a medium containing a mixture of PX+ G.glabra together. These data could be applied for preparation and activation of epididymal and testicular sperms obtained from obstructive azoospermic men.
Background: Unlike sperm production in men which is continuous, women are born with all their eggs and they do not produce any more. Technique to bank eggs would allow girls and women to have the same reproductive options as men when faced with a serious disease and the long term complications of chemotherapies that threatens to destroy their eggs. Objective: The aim of this study is to improve the efficiency of existing ovarian cryopreservation, by following the most recent method named needle immersed vitrification (NIV) beside the slow freezing method. In this review we discussed also the historical description of the methods used, freezing and vitrification of mature and immature oocytes, this include comparison of principles, procedures and results as reported in the literature. Materials and Methods: A total of 285 fragments of human ovarian tissues were isolated and cryopreserved from 36 women aging 26 - 55 years old in the Reproductive Unit, Lubeck-Germany. Ovarian tissue strips of 1-3 mm thick of ovarian cortical biopsies obtained from patients. The collected human ovarian cortex fragments were assigned to two different cryopreservation procedures, slow-freezing and NIV. On the other hand, a total of 80 patients (100 cycles) with polycystic ovaries (PCO) were underwent in vitro maturation (IVM) treatment. The number of inspirated immature oocytes was 536. Vitrification of immature oocytes were carried out. Results: Successful freezing and storing were performed on 285 fragments of ovarian cortex tissue from 36 patients by two cryopreservation protocols as mentioned before but these tissues do not thaw and use yet. Concerning the IVM, the percentage of maturation, fertilization, survival, embryo transfer, and pregnancy rates were 55.2%, 44.5%, 96.2%, 88.1%, and 10.6%, respectively. Conclusion: Recent advances in freezing technology, modifications of conventional protocols used and continuing optimization of vitrification have efficiently improved the method. These results may lead to increase the possibility of preserving fertility by cryopreservation of ovarian tissue. Ovarian tissue banking can offer hope for cancer patients who want to safe guard their fertility against sterilizing chemotherapy and radiotherapy. Immature oocytes can be successfully isolated from the oophorectomy specimen regardless of the day of menstrual cycle, and undergo IVM and cryopreservation.
Background: Many studies has been found that the pentoxifylline (PX) and L-carnitine (LC) as motility stimulants showed a positive effect on the activation of sperm in vitro and improve the forward movement after cryopreservation. Objectives: The present study aim to investigate the possibility of using the motility stimulants(i.ePX,LC and mixture of PX with LC medium) for in vitro sperm activation after cryopreservation of epididymal sperms and their effects in vitro on fertilization rate (FR) and early embryonic development (ED) following in vitro fertilization (IVF) process as a model for obstructive azoospermia in men. Methods: Fifty mature male and Fifty mature female mice, 8-12 weeks old were used. Epididymal sperms were obtained from caudal region and in vitro direct activation technique was done after cryopreservation by using four media namely; PX alone, LC alone, a mixture of PX with LC and Ham's F-12 (as control media). The four media were used for IVF of mature ova and FR with ED were recorded. Results: In vitro activation of epididymal sperms with PX, LC and mixture of PX with LC has shown positive effect on sperm concentration, sperm motility, and grade activity of progressive forward movement after cryopreservation. There was a significant (P<0.05) increase of FR in treated media compared with the control medium after 24 hours of insemination. The study showed that the quality and quantity of embryos. Conclusion: It is concluded from the results of the present study that adding PX, LC and mixture of PX with LC medium to the culture medium of cryopreserved epididymal sperms and IVF leads to an improvement in certain sperm function parameters, supports the FR and early ED rate.
Background: it is important to study a history of the science to appreciate the past as a motive for hard work in the present for better future. In the other hand, the factors affecting pregnancy rate of frozen embryos are yet to be clarified at different embryonic developmental stages. Objectives: to record a brief history of embryo cryopreservation in Iraq and to study many clinical and embryological factors that might affect the pregnancy rate. Subjects, materials and Method: a cross-sectional study of many clinical and em-bryological variable, where120 frozen-thawed embryo transfer cycles enrolled in the study at the period from January 2017 till July 2018. The variables were sta-tistically analyzed first by one factor analysis comparing between pregnant and non-pregnant cases; then by multivariate logistic regression analysis to illustrate main factors without a possible confounder effect. Results: In one factor analysis, the following variables showed a significantly high effect on pregnancy rate (P value <0.001) which are women’s age, women’s weight, number of retrieved oocytes, mature oocytes and good quality embryos, developmental stage, E2 level, and endometrial thickness. While in multivariate logistic regression analysis the women’s weight, the number of retrieved oocytes and good quality embryos were the main factors. Conclusions: it is judiciously to consider these factors while managing infertile couples with embryo cryopreservation programs especially the modifiable fac-tors.
Background: Oocyte vitrification is a promising technique. The choice of appropriate types and concentrations of cryoprotectants is essential for the success of the oocytes vitrification. Objective: This study aimed to investigate the effect of vitrification on viability and morphology of oocytes and to compare the effect of several cryoprotectants on the viability and morphology of oocytes during vitrification and post-thawing. Methods: The sheep ovaries were collected from the local abattoir. Only normal and viable oocytes were included in this study. By using cryotop, immature oocytes that were viable with normal morphology were vitrified with 15% DMSO and 15% EG supplemented with 0.0 M, 0.25M, or 0.5 M of either sucrose or trehalose as control and treated groups. Oocytes viability and morphology were assessed post-aspiration and post-thawing. Results: Fromthe results of the present study, the percentage of post-thawing normal and viable oocyte reported with the use of 0.5M trehalose and EG in vitrification solution (VS) significantly)P<0.05) higher than the percentage of post-thawing normal and viable oocyte reported with use of 0.25 M trehalose and EG. Conclusion: Vitrification is simple technique and easy to perform but it needs some experience to prevent any oocyte loss during vitrification and thawing processing. The use of 0.5 M of either sucrose or trehalose in vitrification solution improves the percentage of post-thawing viable and normal oocytes.
For couples with a variety of male factor disorders, intracytoplasmic sperm injection has made pregnancy and motherhood possible. Two hundred-one males who had non-obstructive azoospermia or severe oligospermia were included in this study. One hundred forty trials of ICSI were done by using fresh sperms (fresh group n=140), while 61 trials of ICSI were done by using cryopreserved sperms (cryopreserved group n=61) to compare the conception rates between fresh and cryopreserved sperms used in intracytoplasmic sperm injection (ICSI). Twenty-five samples from each group were collected by doing a testicular biopsy. Pregnancy rates were higher using cryopreserved sperms as compared to fresh spermatozoa, 39.3% compared to 24.3% in the fresh group ( P .030), respectively. Binary logistic regression analysis showed that the odds ratio of achieving pregnancy with cryopreserved sperms was 2.022 higher than with fresh ( P .030). Cryopreservation has no negative effect on the pregnancy rate. Instead, it might improve the results of ICSI in comparison with a fresh sperm sample.
The estimation of oocyte quality in human in vitro fertilization (IVF) is an important point for the embryologists. Oocyte selection and the identification of the best oocytes may help to limit an overproduction of embryos and to improve the results of oocyte cryopreservation programs. The follicular fluid (FF) can be provided easily during oocyte pick-up and known to represents an optimal source for non-invasive biochemical predictors of oocyte quality. However, till now no substance was found to be used as reliable markers of oocyte competence to fertilization, embryo development and pregnancy. Metabolism and ATP levels within the oocyte and adjacent cumulus cells are associated with quality of oocyte and optimal development of a healthy embryo. Lipid metabolism provides a potent source of energy and its importance during oocyte maturation is being increasingly recognised. Objectives: To determine the effect of lipids (Cholesterol. Triglyceride and HDL) level in serum and follicular fluid in predicting oocyte quality, embryo quality and outcome in patients undergoing intracytoplasmic sperm injection program Patients and Methods: Fifty eight (58) infertile women with an age range 21-41 years old undergoing intracytoplasmic sperm injection cycles were included in this study. Lipids (Cholesterol. TG and HDL.,) levels in serum and follicular fluid were measured by auto analyzer for biochemistry (Flexor El_200) and correlation with oocyte quality, embryo quality and ICSI outcome was done. Results: The results of this study showed no significant (p<0.05) difference in lipid levels in serum and follicular fluid between pregnant and non pregnant females as well as non significant correlation was found between lipid level in serum and follicular fluid in relation to oocyte quality and embryo quality. Conclusions: Serum and follicular fluid lipids are not good predictors of oocyte quality, embryo quality or ICSI outcome in women undergoing IVF/ICSI cycles. Accordingly, these biomarkers would be less reflective of the follicular environment and that the effect of maternal metabolic disorders on oocyte quality and fertility outcome is complex and of a multifactorial kind.