Part Of Assisted Reproductive Technologies Research Articles

Cryopreservation of mammalian oocytes and embryos: current problems and future perspectives.

Cryopreservation techniques for mammalian oocytes and embryos have rapidly progressed during the past two decades, emphasizing their importance in various assisted reproductive technologies. Pregnancies and live births resulting from cryopreserved oocytes and embryos of several species including humans have provided proof of principle and led to the adoption of cryopreservation as an integral part of clinical in vitro fertilization. Considerable progress has been achieved in the development and application of the cryopreservation of mammalian oocytes and embryos, including preservation of the reproductive potential of patients who may become infertile, establishment of cryopreserved oocyte banks, and transport of oocytes and embryos internationally. However, the success rates are still far lower than those obtained with fresh oocytes and embryos, and there are still obstacles that need to be overcome. In this review, we address the major obstacles in the development of effective cryopreservation techniques. Such knowledge may help to eliminate these hurdles by revealing which aspects need improvement. Furthermore, this information may encourage further research by cryobiologists and increase the practical use of cryopreservation as a major part of assisted reproductive technologies for both humans and animal species.

Морфофункціональні характеристики свіжовиділених і кріоконсервованих клітин гранульози та кумулюса яєчників людини

Utilization of granulosa and cumulus cell (GCC) complex could be prospective for co-culture of gametes and embryos as a part of assisted reproductive technologies (ART), that makes important their cryopreservation. The study was performed in GCCs of women aged 25–39, who underwent the treatment of infertility by IVF. Cryopreservation of GCC suspension was carried out using 1.5 M 1,2-propane diol solution. The samples were cooled with rate of 0.3 deg/min from 25 down to –6°C, thereafter the ice crystal initiation was done, from –6 down to –35°C the cooling rate was 1 deg/min, after that the samples were plunged into liquid nitrogen and stored then at –196°C. Following thawing the GCCs retained their main features: ability for adhesion and proliferation, and hormone production. During culture of GCCs its development was delayed for 1–2 days if compared with freshly isolated cells. In 7 days after explanting the cultures of frozen-thawed GCCs appeared nearly the same as primary cultures of corresponding terms of in vitro culture. The post thaw level of hormone production by GCCs was changed: decreased by 89.9% in case of estradiol, and increased by 81.4% for progesterone if compared with the indices of freshly isolated suspensions. The obtained results testify to a possibility to isolate the GCCs and then to store in low-temperature banks for prospective use in ART. Probl Cryobiol Cryomed 2014; 24(1):57–66.

Low laboratory relative humidities appear to affect initial conception rates but not on-going pregnancies in an ART program

It is also well established that well controlled humidity is an essential part of assisted reproductive technologies (ART) culture conditions and necessary for optimum embryo development. However, it is unclear to what extent lab relative humidity (RH), especially low RH, might impact the culture environment. For the last 24 months, environmental conditions in the Southwestern U.S. have produced extended periods of extremely low RH, which has resulted in lower overall building RH at this facility and providing an opportunity to examine the role of lab RH on culture conditions.

The Impact of Ovarian Stimulation on the Expression of Candidate Reprogramming Genes in Mouse Preimplantation Embryos

Ovarian stimulation with gonadotrophins is an integral part of assisted reproductive technologies in human subfertility/infertility treatment. Recent findings have associated ovarian stimulation with the increased incidence of imprinting disorders in humans as well as defects in genome-wide methylation reprogramming and, in particular, imprinting in mice. Here, we present the first study that determined the impact of ovarian stimulation on the expression of developmentally important reprogramming genes (Apex1, Lig1, Lig3, Mbd2, Mbd3, Mbd4, and Polb) in single early mouse morula embryos (16-cell stage). Using absolute quantification of mRNA by quantitative real-time PCR, we observed an association of ovarian stimulation with a downregulation of mRNAs encoding the base excision repair proteins APEX1 and POLB as well as the 5-methyl-CpG-binding domain protein MBD3 in individual morula embryos. Whole mount immunofluorescence staining of early and late morula embryos with an antibody against APEX1 revealed individual embryos with lower protein expression levels after ovarian stimulation and a correlation of mRNA expression with protein abundance. Our data argue for a negative impact of ovarian stimulation during female gametogenesis and/or early embryo development affecting the expression of candidate reprogramming factors.

Studies on reactivation of regressing bonnet monkey corpus luteum on day 1 of menses: A pilot study

Studies on functional characteristics of the regressing primate corpus luteum (CL) to luteotrophic stimulus on day 1 of the non-fertile menstrual cycle are scarce. Recombinant human luteinizing hormone (rhLH) (20 IU/Kg BW; n = 10) or human chorionic gonadotropin (hCG) (180 IU; n = 6) were administered intravenously to female bonnet monkeys on day 1 of menses. Exogenous treatment of rhLH or hCG caused a significant increase in circulating progesterone (P4) levels 2-4 hours post treatment (P < 0.05). Lutectomy prior to onset of menses confirmed that CL is the site of the increased P4 concentrations. Increased levels of phosphorylated P44/42 MAPK, MKK3/6 activation and concomitant histological changes were observed within 4 hours in CL of monkeys receiving hCG treatment. The results from this study demonstrate the acute progesterone synthesizing capacity of regressing monkey CL after LH or hCG challenge. This has potential implications for interpreting the steroidogenic response after gonadotropin stimulation tests in the early follicular phase of the normal ovulatory and anovulatory women undergoing controlled ovarian stimulation protocols as part of assisted reproductive technology (ART) and in women with polycystic ovarian syndrome.

Predicting and preventing ovarian hyperstimulation syndrome (OHSS): the need for individualized not standardized treatment.

Ovarian hyperstimulation syndrome (OHSS) is the most serious complication of controlled ovarian stimulation (COS) as part of assisted reproductive technologies (ART). While the safety and efficacy of ART is well established, physicians should always be aware of the risk of OHSS in patients undergoing COS, as it can be fatal. This article will briefly present the pathophysiology of OHSS, including the key role of vascular endothelial growth factor (VEGF), to provide the foundation for an overview of current techniques for the prevention of OHSS. Risk factors and predictive factors for OHSS will be presented, as recognizing these risk factors and individualizing the COS protocol appropriately is the key to the primary prevention of OHSS, as the benefits and risks of each COS strategy vary among individuals. Individualized COS (iCOS) could effectively eradicate OHSS, and the identification of hormonal, functional and genetic markers of ovarian response will facilitate iCOS. However, if iCOS is not properly applied, various preventive measures can be instituted once COS has begun, including cancelling the cycle, coasting, individualizing the human chorionic gonadotropin trigger dose or using a gonadotropin-releasing hormone (GnRH) agonist (for those using a GnRH antagonist protocol), the use of intravenous fluids at the time of oocyte retrieval, and cryopreserving/vitrifying all embryos for subsequent transfer in an unstimulated cycle. Some of these techniques have been widely adopted, despite the scarcity of data from randomized clinical trials to support their use.

Estimation of the Contribution of Nonassisted Reproductive Technology Ovulation Stimulation Fertility Treatments to US Singleton and Multiple Births

Infertility treatment with ovulation stimulation, either as part of assisted reproductive technologies (ARTs) or for non-ART infertility treatment, has been primarily responsible for the marked increase in multiple births in the United States in recent decades. The multiple-gestations resulting from both ART and non-ART treatments are associated with adverse sequelae, including markedly higher risks of pregnancy complications, premature delivery, infant death, and neurodevelopmental impairment in survivors. A number of studies have reported that ART-conceived singletons are also at higher risk of these outcomes than naturally conceived singletons. Although there is an ongoing, population-based surveillance system in the United States for ART-induced births, there is no comparable system to monitor non-ART infertility treatments, thus the contribution of each to multiple births in the United States is not known. The investigators present data from a multistep modeling process that was developed to estimate the proportions of multiple and singleton livebirths conceived in 2005 by use of non-ART ovulation stimulation. Published surveillance data provided an estimate of the proportion of US multiple births conceived naturally and by ART; it was assumed that the remainder was conceived with non-ART treatments. A meta-analysis of peer-reviewed literature published from 1997 to 2007 was used to estimate the contribution of non-ART ovulation treatment to the risk of multiple-gestation in the annual US birth cohort. A non-ART fetal survival factor was applied to obtain multiple-birth risk estimates. This estimate and the investigator's previous estimate of the proportion of US multiple births attributable to non-ART ovulation stimulation provided an estimate of the total and singleton (derived through subtraction of multiple births) proportion of infants conceived using non-ART induction. Based on the model data, the investigators estimated that the mean percentage of all US births in 2005 conceived with non-ART ovulation stimulation was 4.62% (95% uncertainty range, 2.8%-7.8%), which is 4 times more that the number contributed by ART treatment and equates to more than 190,000 livebirths. Of all singleton pregnancies, 3.98% were conceived with non-ART ovulation stimulation, and 22.8% of all multifetal pregnancies resulted from non-ART ovulation stimulation. These findings demonstrate that non-ART ovulation induction treatment in the United States is associated with a higher multiple-birth-attributable risk than ART. The estimated percentage contribution to US births is 2 to 6 times higher than the contribution of ART.

Challenge testing of gametes to enhance their viability

Embryos, oocytes and spermatozoa undergo several manipulations during the in vitro procedures that are an integral part of assisted reproductive technologies (ART) in mammals. Consequently, some of the gametes are damaged irreparably, whereas others react to these challenges with some sort of survival mechanism that enables them to come through the process. The details of the mechanism remain unknown but, if identified, it could have immense potential as a new way to improve the viability of embryos produced by ART. However, few publications describe systematic ways to challenge test gametes and then to use the results as a basis for improving gamete viability. Furthermore, new methods to monitor the reactions of gametes to such challenge tests are needed. In the present review, these two issues are discussed, as are some of the conditions necessary before a challenge test protocol can be part of future work with ART.

Cryopreserved human blastocysts after vitrification result in excellent implantation and clinical pregnancy rates

We conducted a retrospective review of all frozen ETs from May 2002 to October 2007 using our new vitrification technique in order to determine its efficacy. We found high pregnancy (70.5%; 79/112) and implantation (40.6%; 110/271) rates with the use of this new method.

The Study of Developmental Capacity of Vitrified Mouse Blastocysts in Different Straws after Transfer to Mouse Pseudo Pregnant

Vitrification is the commonly used method for long-term storage of pre-implantation mammalian embryos. It is an essential part of assisted reproductive technologies. The re-expansion rate, pregnancy and birth rate of vitrified blastocysts using CPS were compared with OPS and Conventional Straw. Female NMRI mice were injected with Gonadotrophins in order induce them for super ovulation. At that time the mice were sacrified by cervical dislocation and dissected of mouse abdomen. The uterine horns were existed blastocysts were collected in PBS and randomly allocated to four groups: vitrification in CPS, conventional straw, OPS and untreated controls. The vitrification solution was EFS40%. After storage for 1 month in liquid nitrogen, the blastocysts were thawed in 0.5 M sucrose for in vitro culture in M16 medium. After 6 h of culture, the numbers of expanded blastocysts was recorded and ready for transfer to uterus of pseudo pregnant mouse. The re-expansion rate of the CPS group (72.1%) was significantly higher (p < 0.05) than OPS (52.55) and C.S. (38.6%) groups. The pregnancy (70%) and birth rate (45%) of blastocysts in CPS were similar to those of fresh blastocysts (80% and 45.5%) and the pregnancy (10%) and birth rate (5.1%) in Conventional Straws lower than OPS (20 and 7.5%), but were not significantly different. Mouse blastocysts vitrified using CPS had a better result compared with OPS and Conventional Straw. The value of CPS for vitrification of blastocysts may also merit investigation.

&lt;b&gt;Cryopreservation of Mammalian Embryos: The State of the Art&lt;/b&gt;

Moore K, Bonilla AQ. Cryopreservation of Mammalian Embryos: the State of the Art. ARBS Annu Rev Biomed Sci 2006;8:19-32. Embryo cryopreservation has been a useful tool for embryology since the early 1970s. It has become an essential part of assisted reproductive technologies, allowing long term storage of valuable embryos from lab animals, livestock, endangered species and humans. Initially, work was done with conventional slow cooling technologies, using cryoprotectants, such as glycerol or ethylene glycol and sucrose. More recently, however, greater advances have been made with vitrification, a procedure that bypasses ice crystal formation and places the embryo in a glass-like state. This technology is receiving greater attention as it is simpler, faster and less expensive, and provides the embryo with greater protection from cryoinjury. Successful cryopreservation requires a good understanding of the proper use of cryoprotectants, sugars, and macromolecules in order to allow the highest survival rates and ultimately pregnancy and live offspring following embryo transfer. Although this technology has been in existence for over three decades, it still yields variable results. It is clear now that success is dependent upon many variables, including embryo stage and quality, embryo species and derivation, cooling and warming rates and culture conditions, to name a few. Efforts to optimize these conditions will further enhance survival and future developmental potential of the embryo. This review will briefly summarize the current understanding of embryo cryopreservation technologies, including key areas of concern and strategies to achieve the greatest successes, as well as possible future directions for this field.

Does the Addition of Luteinizing Hormone in Ovarian Stimulation Protocols Improve the Outcome?

Ovarian stimulation is an integral part of assisted reproductive technologies (ART). Under physiologic conditions, both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) activity is necessary to guarantee follicle growth and maturation. This can be shown in patients with hypogonadotrophic hypogonadism, who have no endogenous FSH or LH activity. The use of FSH alone in these patients does not result in sufficient follicle growth and oocyte quality. Approximately 75IU of LH activity per day is necessary to guarantee optimal success. The use of gonadotropin-releasing hormone (GnRH) agonists in normogonadotrophic patients may result in suppression of LH levels below a certain threshold, resulting in suboptimal outcomes. The question under discussion in this article is the threshold level of LH below which exogenous LH activity should be added to provide optimal ovarian response. Different studies indicate that the endogenous LH level should be 0.5-1.5IU in long-term protocol situations. Patients treated in ultra-long GnRH agonist protocols, as well as older patients, patients with a low response to gonadotropin treatment, and patients treated with a GnRH antagonist protocol may benefit from exogenous LH activity. There are three ways of adding LH activity in ovarian stimulation cycles. Nowadays, lutropin alfa (recombinant LH) may be the optimal choice since it has no chorionic gonadotropin activity and allows individual dosage titration. Every menotropin preparation currently on the market contains some chorionic gonadotropin activity. However, more data are necessary before evidence-based recommendations regarding LH supplementation in ovarian stimulation protocols can be given.