The impact of laser-assisted hatching on the outcome of frozen human embryo transfer cycles.

Biochemical modifications of zona pellucida (ZP) result in zona hardening. Zona hardening (ZH) is induced by several factors such as advancing maternal age, in vitro culture conditions and cryopreservation and adversely effects implantation. The objective of the clinical study was to determine whether or not laser-assisted hatching (LAH) applied on day 3 frozen embryos improves the outcome of frozen embryo transfer (FET) cycles in patients with recurrent implantation failure and/or advanced female age. In total, 413 patients of different ages with recurrent implantation failure (maximum three cycles) were involved into the study. Patients were allocated randomly into LAH and control groups. On the day of FET, after thawing and just before FET, the ZP was thinned using a laser system. In the control group no treatment was applied on frozen embryo before transfer. The main outcome measures were clinical pregnancy rate. Overall, the results indicate a tendency that LAH increased (P = 0.08) clinical pregnancy. However, for patients older than 37 years, LAH increased pregnancy rates significantly (P = 0.03). In the LAH and control groups, the age of patients and the number of transferred embryos influenced pregnancy rates (P = 0.01). For patients older than 37 years, no effect of number of transferred embryos was detected (P = 0.14). The incidence of multiple pregnancies also increased in the LAH group (P = 0.01). In conclusion, in older woman, to overcome the negative effect of zona hardening, LAH could be performed on frozen embryos as a routine strategy before FET in frozen cycles in order to increase the possibility of pregnancy formation.

Freezing of oocytes and its effect on the displacement of the meiotic spindle: short communication.

Our investigations focused on spindle dynamics/displacement in frozen-thawed human oocytes. In each oocyte, prior to freezing and after thawing and culturing, the presence/location of the spindle was determined with the Polscope technique. A total of 259 oocytes have been thawed with a survival rate of 81.1%. From the 210 survived oocytes, 165 were fertilized (78.6%) and 89.1% of them cleaved. A total of 143 embryos were transferred into 63 patients resulting in 11 clinical pregnancies (17.5%), 7 of which resulted in live birth of 8 babies (1 twin pregnancy). We were able to detect the spindle in 221 of 259 oocytes (85.3%). After thawing and culturing the oocytes, we were able to visualize the spindle in 177 of 210 oocytes (84.3%). In 83 of these 177 oocytes, the spindle was observed to be in the same location as it was before cryopreservation (46.9%). However, in 94 of these 177 oocytes (53.1%), the spindle reformed in a different position/location relative to the polar body. Our results show that after thawing and culture in half of the spindle-positive oocytes the spindle was detected in a new location, indicating that the spindle and the polar body move relative to each other.

Pregnancy rates with recombinant versus urinary human chorionic gonadotropin in in vitro fertilization: an observational study.

Randomized clinical trials (RCTs) demonstrated the equal efficacy of urinary human chorionic gonadotropin (uhCG) and recombinant hCG (rhCG) products in in vitro fertilisation (IVF). However, limitations inherent with RCTs necessitate the reinforcement of RCT results in real-life. We retrospectively analyzed pregnancies after treatment with rhCG and uhCG products (n = 391, and 96, resp.). We found that laboratory-verified pregnancy occurred more frequently in rhCG patients than in those on uhCG (43% versus 30%, P = 0.02). The association remains significant (P = 0.002) after its adjustment for clinical characteristics. The prevalence of laboratory-verified pregnancies was higher with GnRH agonist use (P = 0.012) and BMI under 30 kg/m(2) (P = 0.053) while decreased the age (P = 0.014) and the number of previous failed attempts (P = 0.08). Similar (but not significant) trends were observed with rates of pregnancy filled the 24th week. These results reinforce RCTs supporting the notion that rhCG is more efficient as uhCG during IVF.

Vitrification of cleavage stage mouse embryos by the cryoloop procedure.

By decreasing the volume of the cryoprotective solution it is possible to increase dramatically the freezing speed and - at the same time - reduce the toxicity and osmotic side effects of cryoprotectants (CPA). The objective of our study was to vitrify Day-3 cleavage stage mouse embryos (n = 229) with the cryoloop technology using a new composition of vitrification media. Embryos were exposed to a 2-step loading of CPA, ethylene glycol (EG) and propylene glycol (PG), before being placed on the surface of a thin filmy layer formed from the vitrification solution in a small nylon loop, then they were rapidly submerged into liquid nitrogen. After warming, the CPA was diluted out from the embryos by a 3-step procedure. Survival of embryos was based on morphological appearance after thawing and continued development to expanded blastocysts upon subsequent 48-hour culture. Embryos of the two control groups were either treated likewise except that they were not vitrified, or cultured in vitro without any treatment. Our data show that a high percentage of embryos survived (92.7%) vitrification in the mixture of EG and PG combined with cryoloop carrier and developed normally (89.1%) in vitro after thawing. To our knowledge this is the first report of the successful vitrification of cleavage stage mouse embryos using VitroLoop vitrification procedure.

The effect of condition/state of testicular spermatozoa injected to the outcome of TESE-ICSI-ET cycles.

OBJECTIVE: The effect of state/condition of spermatozoa (fresh/motile, fresh/immotile, frozen/motile and frozen/immotile) to fertilization, embryo formation/development, implantation and pregnancy/delivery and abortion rates were studied. STUDY DESIGN: The data of a total of 167 TESE-ICSI-ET cycles with fresh and cryopreserved, motile and immotile testicular spermatozoa collected with testicular biopsy from patients suffering from non-obstructive azoospermia were analyzed retrospectively. Analysis of variance (ANOVA) was used to distinguish the group effects in fertilization, embryo formation, and implantation ratio. The group effect was evaluated by using non-parametric statistics and the independent grouping variable was also the "semen state/condition". "Semen state/condition" groups were created according to fresh or frozen, and motile or non-motile (immotile) characteristics. For comparing the four groups, Kruskal-Wallis ANOVA and Median-test was applied. The analysis was carried out using Statistica for Windows (StatSoft, Inc., Chicago, USA). RESULTS: Independently of state/condition of testicular spermatozoa injected into oocytes, no differences were found in fertilization and implantation/pregnancy rates. No difference was obtained in embryo development of oocytes injected with fresh/immotile or frozen/motile spermatozoa. However, difference was found in embryo development of oocytes injected with fresh/motile or frozen/immotile testicular spermatozoa (87% vs. 73%; P<0.04). Comparing embryo development of oocytes injected with fresh vs. frozen spermatozoa difference was also found (83% vs. 74%; P<0.01). No difference was found in the abortion rates between the groups. Differences were observed in the implantation rates, however, these differences could not be verified statistically. CONCLUSION: The presented data show that condition of injected testicular spermatozoa has influence to embryo development and even frozen/immotile testicular spermatozoa is able to induce/support fertilization and early embryo development.

[Deep freezing of oocytes for preserving the fertility of young female oncology patients treated with aggressive radio- and/or chemotherapy]. / A petesejtmélyhutés jelentosége a radio- es/vagy kemoterápiás gyógykezelésben részesített fiatal nobetegek termékenységének megorzésében.

The aggressive radiotherapy and chemotherapy used for treatment of oncological patients--through the damage of germ cells--may cause decrease of fertility or complete infertility. There are relatively few effective clinical options for preserving female fertility. The results of clinical experiments connected with ovarian tissue cryopreservation and oocyte freezing are very promising and indicate that we may have tools for female fertility preservation in the future. In this paper, 1) the future role of oocyte cryopreservation in fertility preservation of female oncological patients, and 2) the authors' preliminary results with oocyte cryopreservation are summarized.

Significant differences in capillary electrophoretic patterns of follicular fluids and sera from women pre-treated for in vitro fertilization.

Human ovarian follicular fluids and sera obtained from women pre-treated for in vitro fertilization (IVF) were investigated by capillary zone electrophoresis. Comparison of the matching physiological liquids showed substantial differences in the electrophoretic patterns. Significant decrease in the alpha(1)- and gamma-fractions of follicular fluids of every woman were observed, whereas other fractions of the samples did not show such alterations. Since follicular fluid is a product of both, secretion by granulosa cells and diffusion from the theca capillaries, we can assume that the forced production of follicular fluid upon hormone stimulation (with gonadotropin releasing hormone (GnRH), follicle stimulating hormone (FSH) and corionic gonadotroph hormone (hCG)) may play role in the uneven presence of the proteins.

Assisted reproductive research: laser assisted hatching and spindle detection (spindle view technique).

Animal experiments are very important for the development of new assisted reproductive techniques (ART) for use in human and animal reproductive medicine. Most technical aspects of reproductive manipulation of humans and animals are very similar, and many components of successful human ART used nowadays have been derived from animal studies. In this study we examined (1) the use of 'non-contact' laser for assisted hatching, (2) whether spindles in living mouse oocytes could safely be imaged/examined by polarisation microscope (polscope) and (3) the influence of environment (e.g. temperature, in vitro culture, etc.) on spindle detection/visualisation. The data of the study presented here show that (1) laser assisted hatching (AH) is a fast, very accurate and safe procedure without any harmful effect on embryo development and it can support very effectively the implantation of embryos, (2) the use of polscope facilitates the evaluation of oocyte quality and the selection of oocytes with spindle, (3) by monitoring the spindle position during intracytoplasmic sperm injection (ICSI), we can reduce spindle damage and increase the chance of fertilisation. Further studies are underway to test the hypothesised connection between spindle birefringence and developmental capacity of oocytes/embryos.

Cryopreservation of embryos and oocytes in human assisted reproduction.

Both sperm and embryo cryopreservation have become routine procedures in human assisted reproduction and oocyte cryopreservation is being introduced into clinical practice and is getting more and more widely used. Embryo cryopreservation has decreased the number of fresh embryo transfers and maximized the effectiveness of the IVF cycle. The data shows that women who had transfers of fresh and frozen embryos obtained 8% additional births by using their cryopreserved embryos. Oocyte cryopreservation offers more advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation, and postponing childbirth, and eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In this review, the basic principles, methodology, and practical experiences as well as safety and other aspects concerning slow cooling and ultrarapid cooling (vitrification) of human embryos and oocytes are summarized.

Birth and clinical pregnancy from fresh and frozen oocytes fertilized with cryopreserved testicular spermatozoa.

This is the first report showing a second clinical pregnancy of a couple who already have a baby from a previous frozen embryo transfer cycle when the embryos were generated from fresh oocytes that were fertilized by intracytoplasmic sperm injection (ICSI) using frozen testicular spermatozoa (the couple have unsuccessful fresh and frozen embryo transfer cycles). Fifty-two months after the first IVF/ICSI cycle the couple had their second IVF/ICSI cycle, but the collected oocytes (n=8) were frozen because no spermatozoa was obtained from the frozen testicular tissue samples which were cryopreserved prior to the first IVF/ICSI cycle. New testicular tissue samples were obtained and frozen. Finally, 58 months after the first IVF/ICSI cycle all of the 8 frozen oocytes of the couple were thawed and fertilized by ICSI using frozen testicular spermatozoa obtained from the newly cryopreserved testicular tissue. Three embryos were transferred and the couple has an ongoing pregnancy, which is in the 20(th) week of pregnancy. Our case report shows that: 1) developmentally competent embryos can be generated by ICSI of frozen-thawed testicular spermatozoa into both fresh and frozen human oocytes, and 2) clinical pregnancy and a healthy baby can be conceived from both frozen and fresh oocytes fertilized with cryopreserved testicular spermatozoa.

The effect of cycle regimen used for endometrium preparation on the outcome of day 3 frozen embryo transfer cycle.

Three cycle regimens to prepare the patients' endometrium for day 3 frozen embryo transfer cycle have been evaluated (natural cycle, hormonally manipulated artificial programmed, and stimulated cycles). All three procedures were equally effective in terms of pregnancy outcome, although a higher probability of pregnancy was found in the natural cycle.

Oocyte cryopreservation: the birth of the first Hungarian babies from frozen oocytes.

PURPOSE: To present data obtained with clinical application of oocyte cryopreservation. METHODS: Slow freezing/rapid thawing in PBS based medium containing 1.5 M propanediol + 0.3 M sucrose. RESULTS: A total of 127 embryos were transferred into 54 patients (1.9 embryo/cycle, 64 transfer cycles). Clinical pregnancy rate of 20% per cycle (13/64) and 24.0% per patient were achieved. Up-to-date, six patients delivered seven healthy babies; there are four ongoing pregnancies. Three abortions (23%) and one biochemical pregnancy (0.7%) was obtained. Implantation rates of 11% per transferred embryos (14/127) and 6.5% (14/215) per thawed eggs were found. In each case, normal karyotype was detected. No difference was found in the ratio of spindle positive oocytes at the polscope analyses done before and after freezing (75.8% vs. 82.5%). CONCLUSION: Egg freezing is not a routine procedure yet, but there will certainly be a place for it in the future of assisted reproductive medicine.

Births resulting from oocyte cryopreservation using a slow freezing protocol with propanediol and sucrose.

The human mature oocyte is particularly sensitive to cooling and low temperatures in addition to freeze-thaw damage. The efficiency of oocyte cryopreservation including the pregnancy outcome is still low. The aim of our study is to briefly introduce our preliminary clinical results achieved with oocyte cryopreservation (CP). Our work focused on the use of a slow cooling procedure using the cryoprotectants propanediol (1.5 M) and sucrose (0.3 M). Following a short incubation of 4-6 hours thawed oocytes were injected with a single sperm (ICSI) and fertilization was assessed 12-16 hours later. Laser assisted hatching (LAH) was performed on all transferred embryos and embryo transfer (ET) was carried out 48-72 hours after ICSI. One-hundred and ten eggs were thawed and a survival rate of 76% (84/110) was obtained. Of the 84 oocytes which survived, 64 subsequently fertilized (64/84; 76%) following ICSI and on the following day 55 of those had cleaved (55/64; 86%). Fifty-two embryos were transferred in 29 patients (1.8 embryo/patient), and 7 (7/29; 24%) resulted in clinical pregnancy (1 twin pregnancy). One of the pregnancies encountered first trimester abortion (1/7; 14%). Implantation rate of 15.4% per embryo transferred (8/52) and 7.3% per egg thawed (8/110) were obtained. In all cases, chorion biopsy was performed and chromosomal anomalities were not detected. Our results provide further evidence that the procedure can be applied safely and with good success in clinical assisted reproduction. However, more work is needed since the survival and implantation rate should be improved.

Does oocyte cryopreservation have a future in Hungary?

The Committee of Human Reproduction established by the Hungarian Ministry of Health is currently working on a proposition that has the intention to ban oocyte cryopreservation in Hungary temporarily. According to the notion of the committee, oocyte cryopreservation and the utilization of frozen oocytes entail enormous risks for future generations. They argue that the safety of the method is unproven. Consequently, their standpoint maintains that cryopreservation invariably remains a grave risk factor with regards to the genetic material of the oocyte by significantly increasing the risk to the offspring. Therefore,the Committee is requesting additional animal experiments to clarify the probability of permanent lesions to the spindle and alterations to the genetic material. According to their point of view, the suspension of the method is further justified since the efficiency of the procedure has yet not reached the level required for clinical adaptation. With the proposed forthcoming step,this commentary takes the opportunity to make critical comments and clarify necessary questions.

Deliveries from embryos fertilized with spermatozoa obtained from cryopreserved testicular tissue.

AIM: The data of 167 TESE-ICSI-ET cycles performed with fresh or frozen, motile or immotile testicular spermatozoa were analyzed, retrospectively. METHODS: The outcome measures studied were state/condition of spermatozoa, fertilization, embryo developmental, implantation and pregnancy/delivery and abortion rates. RESULTS: No differences were found in fertilization, implantation and pregnancy rates of oocytes injected with fresh or frozen spermatozoa. However, difference was obtained in the fertilization rate of oocytes injected with motile vs. non-motile spermatozoa (72% vs. 62%; P < 0.04). Difference was also observed in embryo development between oocytes injected with fresh vs. frozen spermatozoa (83% vs. 75%; P < 0.03). But, no difference was obtained in embryo development between oocytes injected with motile vs. immotile spermatozoa. No difference was also found in the implantation rate of embryos developed from oocytes injected with motile vs. non-motile spermatozoa. No difference was found in abortion rates either. CONCLUSIONS: State/condition of injected testicular spermatozoa has impact to fertilization and embryo development. Pregnancy/delivery can be achieved with frozen/immotile spermatozoa.

Clinical experiences of ICSI-ET thawing cycles with embryos cryopreserved at different developmental stages.

PURPOSE: The aim of our study was to analyze factors including survival, implantation and pregnancy rate, patients' age and BMI, abortions and extra uterine pregnancies that might influence the outcome of ICSI-ET thawing cycles. METHODS: A total of 147 cycles with embryos cryopreserved at different developmental stages were retrospectively evaluated. RESULTS: No difference was found in the survival, implantation and pregnancy rates of embryos cryopreserved on Day 2-3 and 5. However, in the pregnant group significantly higher implantation rate was observed with Day 5 blastocysts then with Day 2 or 3 early embryos. We found no difference in the number of abortions and extra uterine pregnancies between fresh and frozen ICSI-ET cycles. Higher BMI was found in the pregnant than in the nonpregnant group. However, the age of patient had no effect on the results. CONCLUSIONS: Developmental stage of embryo and patients' BMI influences the success of ICSI-ET thawing cycles.

Visualization and examination of the meiotic spindle in human oocytes with polscope.

PURPOSE: The effect of detectability/location of meiotic spindle to fertilization of human MII oocytes and outcome of clinical IVF/ICSI-ET cycles was studied. METHODS: The location of spindle relative to polar body positioned to 6 o'clock was detected with polscope prior to ICSI in oocytes. Data of 83 IVF/ICSI-ET cycle, including pregnancy results, and a total of 1033 oocytes examined were analyzed retrospectively. RESULTS: Polar body position does not always accurately predict spindle position. Similar fertilization was observed in the polscope detected and in the control group. The main number of oocytes and spindle positive oocytes collected was higher in the pregnant than in the non-pregnant group. Higher fertilization was found in that group where 2/3-d of the oocytes had detected spindle. The main number of oocytes and spindle positive oocytes obtained decreased with increasing age of patients. CONCLUSIONS: The presence of birefringent spindle predicts higher fertilization and development and greater probability of pregnancy.