Fertility preservation in patients with gynaecologic malignancy: Response to ovarian stimulation and long-term outcomes.

OBJECTIVE: To the best of our knowledge, the available evidence on the effect and efficacy of controlled ovarian stimulation (COS) in this group of patients remains poorly reported. Concerns related to the impact of stimulation to cancer progression and recurrence, as well as the risk of disease dissemination during egg collection, might explain the aforementioned trend. METHODS: Overall, our FP Service received 192 gynaecological referrals, between 2005 and 2021, regarding gynaecologic conditions mainly cancer related. A total of 68 (35.4%) patients underwent COS. These patients were diagnosed with the following gynaecologic pathologies: 33 cases (48,5%) of cervical cancer were noted (stage 1b1-2b), 25 ovarian pathology (36.7%), 9 cases (13.2%) of endometrial cancer, and a single case of vaginal cancer (1.5%). RESULTS: The mean age of patients attending the fertility preservation service was 31.5 (std 5.8). The patients presenting to their initial appointment with a mean BMI 24.5 (IQR 6.9) and a median AFC of 12 (IQR 13). The mean duration of COS was 11 days (IQR 3), and the median dose of gonadotrophins was calculated at 300 IU (IQR 75 IU). In 95.4% of the cases, GnRH agonist was used as a trigger for final maturation. The median number of follicles measuring more than 14 mm at the time of trigger was 11 (IQR 8), whereas the median number of oocytes collected was 11 (IQR 9). The complication rate was reported at less than 2%. So far, one in four women of this FP group (17/68, 25% of the overall group) returned to our service to claim their cryopreserved eggs/embryos and successful livebirths were reported in 58.8% of this sample (10/17 cases). The mean time to return to use their oocytes/embryos was 36 months (min value 16 months - max value 85 months). There was no significant difference in mortality rate between patients who received FP vs those who did not (hazard ratio of mortality was estimated at 0.91 (p-value 0.88)). CONCLUSION: Based on our findings, ovarian stimulation for patients presenting with gynaecologic malignancy is a safe and efficient method of fertility preservation. Undoubtedly, the sample size is limited, however our results are reassuring and highlight the efficacy of COS for the purpose of FP based on data coming from the largest Assisted Conception Unit of the South-East of the UK.

Outcomes of Social Egg Freezing: A Cohort Study and a Comprehensive Literature Review.

The purpose of this study is to evaluate the live birth outcome following oocyte thaw in women who underwent social egg freezing at Guy's Hospital, alongside a detailed published literature review to compare published results with the current study. A retrospective cohort study was conducted between January 2016 and March 2022 for all women who underwent egg freezing during this period. Overall, 167 women had 184 social egg freezing cycles. The mean age at freeze was 37.1 years and an average of 9.5 eggs were frozen per retrieval. In total, 16% of the women returned to use their frozen eggs. The mean egg thaw survival rate post egg thaw was 74%. The mean egg fertilisation rate was 67%. The pregnancy rate achieved per embryo transfer was 48% and the live birth rate per embryo transfer was 35%. We also noted that irrespective of age at freezing, a significantly high live birth rate was achieved when the number of eggs frozen per patient was 15 or more. Despite the rapid increase in social egg freezing cycles, the utilisation rate remains low. Pregnancy and live birth rate post thaw are encouraging if eggs are frozen at a younger age and if 15 eggs or more were frozen per patient.

Comparing endometrial preparation methods in frozen embryo transfers - Does a previous live birth make a difference?

RESEARCH QUESTION: Does the outcome of a medicated or natural endometrial preparation for a frozen cycle differ if a patient has previously experienced a failed fresh cycle? DESIGN: Retrospective matched case-controlled study to investigate frozen embryo transfer (FET) outcomes in women undergone medicated or natural endometrial preparation, with adjustment to the history of previous live birth. 878 frozen cycles were included for analysis, over a period of 2 years. RESULTS: After adjusting for the number of embryos transferred, endometrial thickness and the number of previous embryo transfers, there was no difference in live birth rate (LBR) between medicated-FET and natural-FET groups regardless of the previous fertility outcome (p = 0.08). CONCLUSIONS: A previous live birth does not affect the outcome of a subsequent frozen cycle, regardless of whether medicated- or natural endometrial preparation is used.

Protocol for developing a core outcome set for male infertility research: an international consensus development study.

STUDY QUESTION: We aim to develop, disseminate and implement a minimum data set, known as a core outcome set, for future male infertility research. WHAT IS KNOWN ALREADY: Research into male infertility can be challenging to design, conduct and report. Evidence from randomized trials can be difficult to interpret and of limited ability to inform clinical practice for numerous reasons. These may include complex issues, such as variation in outcome measures and outcome reporting bias, as well as failure to consider the perspectives of men and their partners with lived experience of fertility problems. Previously, the Core Outcome Measure for Infertility Trials (COMMIT) initiative, an international consortium of researchers, healthcare professionals and people with fertility problems, has developed a core outcome set for general infertility research. Now, a bespoke core outcome set for male infertility is required to address the unique challenges pertinent to male infertility research. STUDY DESIGN SIZE DURATION: Stakeholders, including healthcare professionals, allied healthcare professionals, scientists, researchers and people with fertility problems, will be invited to participate. Formal consensus science methods will be used, including the modified Delphi method, modified Nominal Group Technique and the National Institutes of Health's consensus development conference. PARTICIPANTS/MATERIALS SETTING METHODS: An international steering group, including the relevant stakeholders outlined above, has been established to guide the development of this core outcome set. Possible core outcomes will be identified by undertaking a systematic review of randomized controlled trials evaluating potential treatments for male factor infertility. These outcomes will be entered into a modified Delphi method. Repeated reflection and re-scoring should promote convergence towards consensus outcomes, which will be prioritized during a consensus development meeting to identify a final core outcome set. We will establish standardized definitions and recommend high-quality measurement instruments for individual core outcomes. STUDY FUNDING/COMPETING INTERESTS: This work has been supported by the Urology Foundation small project award, 2021. C.L.R.B. is the recipient of a BMGF grant and received consultancy fees from Exscentia and Exceed sperm testing, paid to the University of Dundee and speaking fees or honoraria paid personally by Ferring, Copper Surgical and RBMO. S.B. received royalties from Cambridge University Press, Speaker honoraria for Obstetrical and Gynaecological Society of Singapore, Merk SMART Masterclass and Merk FERRING Forum, paid to the University of Aberdeen. Payment for leadership roles within NHS Grampian, previously paid to self, now paid to University of Aberdeen. An Honorarium is received as Editor in Chief of Human Reproduction Open. M.L.E. is an advisor to the companies Hannah and Ro. B.W.M. received an investigator grant from the NHMRC, No: GNT1176437 is a paid consultant for ObsEva and has received research funding from Ferring and Merck. R.R.H. received royalties from Elsevier for a book, consultancy fees from Glyciome, and presentation fees from GryNumber Health and Aytu Bioscience. Aytu Bioscience also funded MiOXYS systems and sensors. Attendance at Fertility 2020 and Roadshow South Africa by Ralf Henkel was funded by LogixX Pharma Ltd. R.R.H. is also Editor in Chief of Andrologia and has been an employee of LogixX Pharma Ltd. since 2020. M.S.K. is an associate editor with Human Reproduction Open. K.Mc.E. received an honoraria for lectures from Bayer and Pharmasure in 2019 and payment for an ESHRE grant review in 2019. His attendance at ESHRE 2019 and AUA 2019 was sponsored by Pharmasure and Bayer, respectively. The remaining authors declare no competing interests. TRIAL REGISTRATION NUMBER: Core Outcome Measures in Effectiveness Trials (COMET) initiative registration No: 1586. Available at www.comet-initiative.org/Studies/Details/1586. TRIAL REGISTRATION DATE: N/A. DATE OF FIRST PATIENT'S ENROLMENT: N/A.

Ovarian hyperstimulation syndrome: cardiac arrest with an unexpected outcome.

We describe the acute deterioration of a 29-year-old undergoing in vitro fertilisation. Late-onset critical ovarian hyperstimulation syndrome triggered a massive pulmonary embolism and subsequent cardiac arrest. While the prognosis was deemed to be poor, the patient made a full recovery. The potential reasons for this are explored.

Peri-operative morbidity and fertility outcome after repeat abdominal myomectomy for large fibroid uterus.

This was a retrospective cohort study evaluating peri-operative morbidity of 66 women who underwent repeat abdominal myomectomy compared with 200 women who had primary myomectomy in the same period, matched for age and uterine size. We report the reproductive outcome of women seeking fertility after repeat myomectomy. More pre-operative GnRH analogues were used and midline abdominal skin incision performed in the repeat myomectomy group. The likelihood of major complication was three times higher in the repeat abdominal myomectomy group (OR 3.0, 95% CI 1.67-5.5, p < .001). There was a significantly longer mean hospital stay (p < .01), higher incidence of bleeding (p < .01) and urinary or wound infection (p < .01) in the repeat abdominal myomectomy group. Of the 47 women who had repeat myomectomy for fertility reasons, six women conceived and two live birth at term (4%). Our study highlights the significant peri-operative morbidity and poor subsequent live birth rate associated with repeat abdominal myomectomy.Impact statementWhat is already known on this subject? Repeat abdominal myomectomy is a major surgical procedure with significant morbidity. However, abdominal myomectomy for large fibroid uterus remains the preferred treatment method for women who wish to preserve fertility. Sufficient evidence related to the peri-operative morbidity and fertility outcome after repeat abdominal myomectomy is lacking.What do the results of this study add? This is the largest study reporting peri-operative morbidity and pregnancy outcome following repeat abdominal myomectomy. Our results highlight the three times increased risk of major complications associated with repeat abdominal myomectomy compared to primary myomectomy with the poor subsequent live birth rate.What are the implications of these findings for clinical practice and/or further research? Our study complements the sparse existing data on the outcome of repeat abdominal myomectomy and underscore the potentially significant peri-operative morbidity and poor subsequent live birth rate associated with the procedure. This information should be used in counselling women with fibroid recurrence after primary myomectomy before they embark on repeat surgery.

Planned and unplanned pregnancies in breast cancer survivors.

BACKGROUND: Nearly 20% of women presenting with breast cancer in the UK are of reproductive age. The aim of our study was to assess fertility outcome in relation to intent to conceive in patients who had completed breast cancer treatment. METHODS: Between July 2011 and December 2013, women, aged less than 43 years at the time of diagnosis, were asked during their follow-up in breast cancer clinic, to complete a questionnaire on intentions to conceive and pregnancy outcome. RESULTS: A total of 175 women completed the questionnaire at a median time of 6 years from time of diagnosis. Their median age at the time of diagnosis was 37 years and at the time of the survey was 43 years. At the time of the survey, 42% (72/175) had completed their family, 41% (72/175) reported that they would like to have children and 4% (7/175) did not wish to have children. Twenty-seven respondents had actively tried to conceive, and of those 13 (48%) had a live birth. There were 12 unintended pregnancies; the majority of which were terminated (58% (7/12)) with only 3 live births. Among those who did not wish to conceive, only 32% (36/111) reported using contraception. CONCLUSION: This is a retrospective study investigating menstrual function, contraceptive methods and fertility outcomes of young breast cancer survivors. This study highlights the need for healthcare professionals to provide long-term contraceptive advice to women who do not wish to conceive.

Ovarian reserve in women with sickle cell disease.

BACKGROUND: It has been proposed that ovarian sickling and/or iron overload in women with sickle cell disease (SCD) could contribute to gonadal dysfunction, but there are very few published studies. We hypothesised that the above phenomena might impair ovarian reserve. METHODS: A total of 50 SCD patients were case-matched by age, ethnicity, and presence of regular cycles (28±5 days) with 73 patients without a known haemoglobinopathy who required anti-Müllerian hormone (AMH) assessment in a gynaecology clinic. SCD patients had AMH levels taken as part of routine care. The patients were case-controlled and matched with patients who had no haemoglobinopathy in a tertiary centre over a period of one year. RESULTS: The mean AMH in the SCD case group was 7.6 pmol/l compared with 13.4 pmol/l in the control group (p<0.001). The AMH distributions were subsequently categorised. This showed that SCD patients had a significantly higher chance of having lower AMH in comparison with the control group (OR 2.6 (CI 1.1-6.5, P = 0.02). The proportion of women with AMH > 20 pmol/l was significantly lower in the SCD group (6%) in comparison with the control group (19%) (P = 0.04). CONCLUSIONS: This is the first study showing that women of reproductive age with SCD are more likely to have a low ovarian reserve at a younger age in comparison with patients with no haemoglobinopathy.

Impact of the outcome of fresh blastocyst transfer on the subsequent frozen-thawed blastocyst transfer cycle.

The objective of this observational study was to assess the influence of the outcome of fresh blastocyst transfer on the success rate of the subsequent sibling frozen-thawed blastocyst transfer (FBT) cycle. In total, 1639 FBT cycles were divided into two groups: Group A (n = 698) cycles in which a positive pregnancy test result was achieved and Group B (n = 941) cycles in which no pregnancy was achieved in the preceding fresh IVF cycle. Mean age at cryopreservation, basal FSH level, number of oocytes retrieved, number of embryos transferred in the fresh cycle and survival rate of the thawed blastocysts in the FBT cycle were comparable between the two groups. Although significantly more thawed blastocysts were transferred in the FBT cycles in Group B compared with Group A, the live birth rate in Group A was significantly higher compared with Group B. After adjusting for potentially confounding variables, the likelihood of a live birth after FBT was significantly higher when a pregnancy was achieved in the preceding fresh IVF cycle. Achieving a pregnancy after fresh blastocyst transfer is an independent factor influencing the outcome of the subsequent sibling FBT.

An update on the prevention of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome is a potentially life-threatening, but preventable iatrogenic complication of in vitro fertilisation treatment. In recent years, new strategies have been developed to minimise the risk of ovarian hyperstimulation syndrome after in vitro fertilisation, including better at-risk patient identification prior to starting treatment, the use of a lower human chorionic gonadotrophin dose or alternative medication instead of human chorionic gonadotrophin to induce final oocyte maturation such as gonadotrophin-releasing hormone agonist and kisspeptin in antagonist cycles, cryopreservation of all embryos and delayed embryo transfer, and the use of oral dopamine agonists after oocyte retrieval. In this article, the advantages and limitations of those new developments are discussed and future directions towards establishment of an ovarian hyperstimulation syndrome-free in vitro fertilisation clinic are explored.

Defining safe criteria to diagnose miscarriage: prospective observational multicentre study.

OBJECTIVES: To validate recent guidance changes by establishing the performance of cut-off values for embryo crown-rump length and mean gestational sac diameter to diagnose miscarriage with high levels of certainty. Secondary aims were to examine the influence of gestational age on interpretation of mean gestational sac diameter and crown-rump length values, determine the optimal intervals between scans and findings on repeat scans that definitively diagnose pregnancy failure.) DESIGN: Prospective multicentre observational trial. SETTING: Seven hospital based early pregnancy assessment units in the United Kingdom. PARTICIPANTS: 2845 women with intrauterine pregnancies of unknown viability included if transvaginal ultrasonography showed an intrauterine pregnancy of uncertain viability. In three hospitals this was initially defined as an empty gestational sac <20 mm mean diameter with or without a visible yolk sac but no embryo, or an embryo with crown-rump length <6 mm with no heartbeat. Following amended guidance in December 2011 this definition changed to a gestational sac size <25 mm or embryo crown-rump length <7 mm. At one unit the definition was extended throughout to include a mean gestational sac diameter <30 mm or embryo crown-rump length <8 mm. MAIN OUTCOME MEASURES: Mean gestational sac diameter, crown-rump length, and presence or absence of embryo heart activity at initial and repeat transvaginal ultrasonography around 7-14 days later. The final outcome was pregnancy viability at 11-14 weeks' gestation. RESULTS: The following indicated a miscarriage at initial scan: mean gestational sac diameter ≥ 25 mm with an empty sac (364/364 specificity: 100%, 95% confidence interval 99.0% to 100%), embryo with crown-rump length ≥ 7 mm without visible embryo heart activity (110/110 specificity: 100%, 96.7% to 100%), mean gestational sac diameter ≥ 18 mm for gestational sacs without an embryo presenting after 70 days' gestation (907/907 specificity: 100%, 99.6% to 100%), embryo with crown-rump length ≥ 3 mm without visible heart activity presenting after 70 days' gestation (87/87 specificity: 100%, 95.8% to 100%). The following were indicative of miscarriage at a repeat scan: initial scan and repeat scan after seven days or more showing an embryo without visible heart activity (103/103 specificity: 100%, 96.5% to 100%), pregnancies without an embryo and mean gestational sac diameter <12 mm where the mean diameter has not doubled after 14 days or more (478/478 specificity: 100%, 99.2% to 100%), pregnancies without an embryo and mean gestational sac diameter ≥ 12 mm showing no embryo heartbeat after seven days or more (150/150 specificity: 100%, 97.6% to 100%). CONCLUSIONS: Recently changed cut-off values of gestational sac and embryo size defining miscarriage are appropriate and not too conservative but do not take into account gestational age. Guidance on timing between scans and expected findings on repeat scans are still too liberal. Protocols for miscarriage diagnosis should be reviewed to account for this evidence to avoid misdiagnosis and the risk of terminating viable pregnancies.

The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence.

BACKGROUND: Cryopreservation has been extensively used in assisted reproductive technology, agriculture and conservation programmes for endangered species. The literature reports largely positive results regarding the survival of frozen-thawed cells and clinical outcomes. Nonetheless, it is unclear whether or not cryopreservation of sperm, oocytes and embryos causes any disruption in their genetic integrity. Drawing on the available published evidence, this review paper describes in detail the physical and biochemical factors of cryopreservation that could potentially affect genomic integrity. METHODS: A critical review of the published literature using PubMed with particular emphasis on studies which include assessment of genetic stability after cryopreservation of oocyte, sperm and embryos. The search was performed in 2014 and covered the period from the beginning of electronic records until July 2014. No language restrictions were applied. RESULTS: Cryopreservation is associated with extensive damage to cell membranes, and results in alteration of the functional and metabolic status of the cells and mitochondria. Some evidence suggests an increase in DNA single-strand breaks, and degree of DNA condensation or fragmentation in sperm after cryopreservation. The extent of these changes may vary between different individuals and different techniques. The addition of antioxidants to the cryopreservation media and the use of well-controlled cooling regimes could potentially improve such outcomes. Limited numbers of studies on oocytes provide controversial results regarding the effect on DNA fragmentation, sister chromatid exchange (SCE) and aneuploidy. The only study on human embryos suggested that vitrification affects DNA integrity to a much lesser extent than slow freezing. Animal studies show increases in mitochondrial DNA mutations in embryos after cryopreservation. The limited numbers of long-term follow-up studies in humans provide reassurance that derives mostly from retrospective studies with some methodological weaknesses. CONCLUSIONS: This review provides an overview of studies performed to date on the effect of cryopreservation on the oocyte, sperm and embryos. Controversy of the reported data has highlighted the gaps in our knowledge not only for clinical studies, but also for basic research in human embryos. New perspectives for future research are proposed.

Impact of obesity on pregnancy outcome in different ethnic groups: calculating population attributable fractions.

OBJECTIVES: To quantify the proportion of adverse pregnancy outcome attributable to maternal obesity. DESIGN: Cross sectional analysis of routine obstetric dataset. SETTING: Guy's and St Thomas's NHS Foundation Trust (GSTFT). POPULATION: 23,668 women who had singleton deliveries at GSTFT between 2004 and 2008. METHODS: Logistic regression was used to estimate the association between BMI and outcome in different ethnic groups. Adjusted odds ratios, and the proportions of obese women, were used to calculate population attributable risk fractions (PAFs). MAIN OUTCOME MEASURES: (I) MATERNAL OUTCOMES: diabetes, type of delivery, post-partum haemorrhage, and preterm delivery. (ii) Perinatal outcomes: macrosomia, low birth weight, admission to neonatal intensive care/special care baby unit, and perinatal death. RESULTS: The prevalence of maternal obesity was 14%. Increasing BMI was independently associated with increasing risk of adverse obstetric and neonatal outcome. At the individual level, the effect of obesity on diabetes was highest in Asian women compared to white women (p for interaction = 0.03). Calculation of population attributable risk fractions demonstrated that one third of diabetes cases and one in six Caesarean sections could be avoided in this population if all obese women were of normal BMI. At the population level, the contribution of obesity to diabetes was highest for Black women (42%), and lowest for oriental women (8%). Seven percent of neonatal macrosomia in all the population, and 13% in Black mothers, were attributable to obesity. CONCLUSIONS: Preventing obesity prior to pregnancy will substantially reduce the burden of obstetric and neonatal morbidity in this population. This reduction will be higher in Black women.

Endocervical crypt involvement by high-grade cervical intraepithelial neoplasia after large loop excision of transformation zone: do we need a different follow-up strategy?

AIMS: The aim of this study was to determine the effect of endocervical crypt involvement in specimens with clear margins on recurrence following large loop excision of transformation zone (LLETZ). MATERIAL AND METHODS: This was an observational cohort study. In the colposcopy unit in a university teaching hospital we prospectively collected data for women who underwent LLETZ treatment for high-grade cervical intraepithelial neoplasia (CIN) between 2003 and 2004. We determined the difference in recurrence rate and need for repeat treatment between groups with and without crypt involvement in the primary histology. We prospectively collected data of follow up until 2010. The recurrence was analyzed using Cox regression. RESULTS: A total of 309 women had complete excision of the margins following LLETZ treatment for CIN 2 or 3. There was no significant difference in age between groups with (30.2) and without (29.7) crypt involvement (P<0.25). There was a significant difference in the prevalence of abnormal smear results before and after LLETZ between groups with or without crypt involvement (P=0.043). The need to perform a repeated treatment was significantly different between groups with and without crypt involvement (P<0.024). A full model significantly predicted recurrence of cervical pathology (P<0.009) that necessitated treatment when crypt involvement was present. The odds ratio for repeat treatment with crypt involvement was 2.67 (confidence interval, 1.27-5.64). CONCLUSIONS: Our study showed that positive involvement of endocervical crypt by CIN in a cervical loop excision specimen increases the frequency of subsequent episodes of treatment.

Does hydrothermal ablation avoid hysterectomy? Long-term follow-up.

OBJECTIVE: We sought to assess the long-term success rate of the HydroThermAblator system (HTA). STUDY DESIGN: We conducted a retrospective cohort study of 376 patients who underwent HTA at our hospital during an 8-year period, following case note review and distribution of a validated menorrhagia questionnaire. RESULTS: The mean age of patients was 43 years. Operative complications included 3 women (0.8%) who experienced intraoperative burns. Of the 248 (66%) returned questionnaires, satisfaction rates were high at 77%. The amenorrhea rate was 38%, with a further 37% of women reporting a substantial decrease in their blood loss. In all, 29 (11%) women underwent subsequent hysterectomy for persistent menorrhagia or dysmenorrhea. Younger women had a significantly higher chance of proceeding to subsequent (P < .05) hysterectomy. CONCLUSION: This study confirms the long-term patient satisfaction with HTA and that the overall probability of proceeding to subsequent hysterectomy over 8 years was only 11%.

Cryopreservation of fish sperm.

This chapter provides detailed descriptions of the protocol used for fish sperm cryopreservation. The principles described can be applied to any species, but details are also given for individual fish species. Successful cryopreservation of fish spermatozoa depends on a range of factors including the collection of high quality sperm, equilibration conditions, choice of cryoprotectant medium, cooling/thawing regimes, and conditions for fertilization. Even though some general rules can be applied to any fish species, optimization of the protocol is needed for each individual species. Furthermore, because sperm derived from marine and freshwater fish differs markedly, the described protocol is mainly focused on the cryopreservation of sperm for freshwater fish because this poses the greater technical challenge.

Zebrafish embryos (Danio rerio) using microinjection.

Low membrane permeability is one of the major obstacles to the successful cryopreservation of zebrafish embryos. The aim of the present study was to explore if this could be overcome by yolk modification with different cryoprotectants by micro-injection. Initial investigation of two cryoprotectants, methanol and sucrose, was undertaken to determine their suitability for micro-injection supplementation of the yolk mass. Intact zebrafish embryos at 50% epiboly stage were injected with Hanks' solution, 5.2 M methanol or 1.3 M sucrose yielding approximate final concentrations of 2.0 and 0.5 M of the cryoprotectants within the yolk sac respectively. After micro-manipulation, the embryos were cultured at 28 degree C for three days and their survival assessed at the hatching stage. All micro-manipulations performed in the present study resulted in a significant decrease in embryo survival (P < 0.05). Embryos micro-injected with methanol or sucrose were also subjected to a cooling procedure. They were placed in 3M methanol + 0.5 M sucrose at room temperature for 30 min and then cooled from 20 degree C to 0 degree C at 2 degree/min, from 0 degree C to -7.5 degree/min at 1 degree/min, seeded at -7.5 degree C and held for 10 min, before cooling at 0.3 degree/min to - 20 degree C or until full crystallization in all embryos. The processes of extra- and intracellular crystallization were studied by cryomicroscopy. The temperature of intracellular crystallization did not differ significantly between control and injected embryos. However, it was found that intracellular crystallization did not always happen instantly after extracellular crystallization.

Effect of cryopreservation on mitochondrial DNA of zebrafish (Danio rerio) blastomere cells.

Cryopreservation has been extensively used in human reproductive medicine, aquaculture and conservation programmes for endangered species. However, despite the growing successes of cryopreservation, post-thaw recovery of reproductive and embryonic cells very often remains poor. Many studies have been devoted to the mechanisms of cryodamage. It is known that cryopreservation causes extensive damage to membranes; reduce the metabolic activity of cells; and disturbs the mitochondrial bioenergetical processes of cells. But few investigations on the genetic stability of cells during cryopreservation have been performed, and the role of any genetic impact cryopreservation needs to be determined. Some indirect data in the literature suggests that progress in this field might come from investigating freezing damage to mitochondrial DNA (mtDNA), nuclear DNA and other genome-related structures. In this study, zebrafish (Danio rerio) blastomeres were treated in three different ways: control suspension of blastomere cells in phosphate buffered saline; equilibration of blastomeres with 2M dimethyl sulfoxide (Me2SO) for 1h at room temperature and cryopreservation using Me2SO as a cryoprotectant. Mitochondrial DNA was analysed in fresh cells and after the different treatments. Two different loci of mtDNA were amplified with the help of PCR and sequenced. The sequences were analysed and nuclear base substitutions were counted for both control and treated samples. The results showed that cryopreservation significantly increased the frequency of mutations (0.78+/-0.27% in comparison to 0.16+/-0.25% of control), whilst 2M Me2SO treatment did not bring a significant increase in frequency of mutations (0.24+/-0.28%). The distributions of the mutation locations were analysed. More investigations are needed to determine whether optimisation of cryopreservation protocol is possible to reduce these adverse effects; whether such mutations interfere with overall function of the cells; whether similar changes also occur in the nuclear DNA and whether such mutations happen in other species. Meanwhile, it is important to be cautious in making judgements of the effect of cryopreservation technique in assisted reproduction. This is the first report on the effect of cryopreservation on mtDNA.

Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm.

Semen cryopreservation is widely used in clinical medicine, agriculture, aquaculture and biomedical research, but it is an inefficient technique that induces extensive cytoplasmic damage and loss of fertilising ability. Whether any genetic damage (i.e. DNA strand breakage or mutation) is also induced is still unclear. However, previous data has indicated that this is likely. The present study was designed to explore this possibility further by using inhibitors of the DNA repair system to block DNA repair in embryos derived from cryopreserved spermatozoa. If cryopreservation causes strand breaks in sperm DNA it might be expected that inhibition of a repair enzyme such as poly(ADP-ribose) polymerase (PARP) would enhance any such negative effect of cryopreservation. To check this hypothesis 3-aminobenzamide (3-AB) was used as an inhibitor of PARP. Weather loach (Misgurnus fossilis) eggs were fertilised using cryopreserved as well as fresh spermatozoa. Embryos derived from cryopreserved spermatozoa were exposed to 10 mM 3-AB for 2 h after fertilisation. The experiments were carried out using 43,544 embryos from 5 females and 10 males. Embryo survival was evaluated at different stages until the hatching stage. Sperm cryopreservation significantly decreased embryo survival (53.6+/-2.79% compared to 76.97+/-2.79% of control; P<0.01). The addition of 3-AB to the medium with embryos derived from cryopreserved sperm further decreased embryo survival from 53.6+/-2.79% to 46.1+/-2.79% (P<0.01) whereas there was no adverse effect of 3-AB exposed embryos derived from fresh sperm (76.97+/-2.79% of control compared to 74.8+/-2.79% of control+3-AB). The effect of 3-AB provides indirect evidence that cryopreservation might induce instability in sperm DNA, and that such damage can be repaired by the oocyte repair system after fertilisation.