Nina Desai

Slow freezing, vitrification and ultra-rapid freezing of human embryos: a systematic review and meta-analysis

Embryo cryopreservation is an important aspect of assisted reproduction. Many methods have been described, but they have been poorly investigated in randomized trials, highlighting the need for a systematic review of the literature. Meticulous electronic/hand searches were performed to locate randomized trials (RCT) comparing embryo cryopreservation methods. Primary outcomes were clinical pregnancy rate (CPR) and incidence of congenital abnormalities. Secondary outcomes included live-birth (LBR), ongoing pregnancy (OPR), implantation (IR), and miscarriage (MR) rates. Data were extracted to allow for an intention-to-treat analysis and analysed using a random-effects model. Literature search revealed 11 RCT, of which five were excluded. The quality of the included studies was variable, but generally poor. There was a significantly higher CPR, OPR and IR with vitrification compared with slow freezing (odds ratio (OR)=1.55, 95% confidence interval (CI)=1.03-2.32, OR=1.82, 95% CI=1.04-3.20 and OR=1.49, 95% CI=1.03-2.15, respectively). In addition, there was a significantly lower CPR and OPR with embryo ultra-rapid freezing compared with slow freezing (OR=0.35, 95% CI=0.16-0.76 and OR=0.37, 95% CI=0.17-0.81, respectively). Vitrification is superior to slow freezing, which in turn is superior to ultra-rapid freezing. However, more well-designed and powered studies are needed to further corroborate these findings.

Gonadotropin-releasing hormone analog cotreatment for preservation of ovarian function during gonadotoxic chemotherapy: a systematic review and meta-analysis.

OBJECTIVE To determine whether gonadotropin-releasing hormone (GnRH) analog cotreatment with chemotherapy provides better reproductive outcomes for women at risk of premature ovarian failure (POF) as a side-effect of gonadotoxic chemotherapy. DESIGN Systematic review and meta-analysis. SETTING University-affiliated research centers. PATIENT(S) None. INTERVENTION(S) Electronic and manual searches (e.g., MEDLINE, EMBASE, CENTRAL) up to January 2010 were performed to identify randomized controlled trials (RCTs) comparing GnRH cotreatment with chemotherapy alone in premenopausal women. MAIN OUTCOME MEASURE(S) Incidence of POF after treatment, incidence of women with resumption of ovulation, POF after an initial normal cycle, normal cycles but abnormal markers of ovarian reserve, spontaneous occurrence of pregnancy after treatment, and time to reestablishment of menstruation; data also extracted to allow for an intention-to-treat analysis. RESULT(S) Twenty-eight RCTs were identified, but only six met the inclusion criteria. Data were only available for the incidence of women with new onset of POF, resumption of ovulation, and occurrence of pregnancy. The incidence of POF or resumption of ovulation both demonstrated a statistically significant difference in favor of the GnRH cotreatment. The occurrence of spontaneous pregnancy showed no statistically significant difference between GnRH cotreatment and the control groups. CONCLUSION(S) Evidence from RCTs suggests a potential benefit of GnRH cotreatment with chemotherapy in premenopausal women, with higher rates of spontaneous resumption of menses and ovulation but not improvement in pregnancy rates. Data relating to study quality and possible bias for the majority of the outcomes in this review were not available, denoting possible selective reporting of trial data.

Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births

Vitrification technology has shown great promise for cryopreservation of human embryos. The majority of this work has been with blastocyst-stage embryos. This report describes initial clinical results following vitrification of human embryos on day 3 of culture at the 6- to 8-cell stage. A total of 236 embryos were cryopreserved on cryoloops using a vitrification protocol. Warmed embryos were cultured until day 5 before transfer to the patient. The post-warming survival rate was 85%. The clinical pregnancy rate was 44% (34/77), and the implantation rate was 20% (40/201). In transfers where at least one warmed embryo reached the blastocyst stage by the day of transfer, the clinical pregnancy rate was 58% (28/48). The cryoloop was an excellent vessel for ultra-rapid cryopreservation of embryos. This study supports the effectiveness of a dimethylsulphoxide/ethylene glycol cryoprotectant combination for vitrification of human embryos at the 6- to 8-cell stage.

Reproductive outcome after transplantation of ovarian tissue: a systematic review

BACKGROUND Despite interest in ovarian tissue transplantation (OTT) as a promising procedure for fertility preservation, to date, no precise data are available about its effectiveness. We systematically reviewed reproductive function after OTT for fertility preservation in women at high risk of premature ovarian failure (POF). METHODS We searched the MEDLINE, EMBASE, Cochrane Systematic Reviews, CENTRAL, Web of Science and Scopus databases for studies on the reproductive outcomes after OTT in humans up to June 2007. Women with follicle-stimulating hormone (FSH) >30 IU/l at the time of OTT were included in a meta-analysis of individual-patient data to evaluate the time to re-establishment of ovarian function (ROF). Secondary outcomes included short-term (<12 months) and long-term (>12 months) ovarian function (OVF) and pregnancy after OTT. RESULTS We identified 25 reports including 46 unique cases. OTT was performed to treat POF in 27 women, to prevent POF in 15, to treat infertility in 2 and accidentally in 1. In 23 women with FSH >30 at the time of OTT, OVF was re-established with a median time to ROF of 120 days (range 60-244). Within 6 months after ROF, four women had recurrent ovarian failure. There are insufficient data to evaluate the long-term OVF (>12 months). Fresh grafts had an increased likelihood of return of OVF and a decreased likelihood for recurrent ovarian failure compared with cryopreserved grafts [HR of 2.44 (95% CI 0.92, 6.49) and 0.47 (95% CI 0.18, 1.12), respectively]. In 25 women who sought pregnancy, eight women had nine pregnancies at 12 months, giving a cumulative pregnancy rate of 37% (95% CI 19, 60). CONCLUSIONS Transplantation of ovarian tissue can re-establish OVF after POF; however, the efficacy of OTT using cryopreserved tissues is not yet equivalent to that of fresh grafts. A controlled multicenter trial with sufficient follow-up would provide valid evidence of the potential benefit of this procedure.

Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles

BackgroundTime-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting.MethodsKinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed.ResultsA total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation and reverse cleavage amongst the embryos observed was 25% and 7%, respectively. Over 40% of embryos exhibiting these characteristics did however form blastocysts meeting our criteria for freezing.ConclusionsThese data provide us with a platform with which to potentially enhance embryo selection for transfer.

Three-dimensional in vitro follicle growth: overview of culture models, biomaterials, design parameters and future directions

In vitro ovarian follicle culture is a new frontier in assisted reproductive technology with tremendous potential, especially for fertility preservation. Folliculogenesis within the ovary is a complex process requiring interaction between somatic cell components and the oocyte. Conventional two-dimensional culture on tissue culture substrata impedes spherical growth and preservation of the spatial arrangements between oocyte and surrounding granulosa cells. Granulosa cell attachment and migration can leave the oocyte naked and unable to complete the maturation process. Recognition of the importance of spatial arrangements between cells has spurred research in to three-dimensional culture system. Such systems may be vital when dealing with human primordial follicles that may require as long as three months in culture. In the present work we review pertinent aspects of in vitro follicle maturation, with an emphasis on tissue-engineering solutions for maintaining the follicular unit during the culture interval. We focus primarily on presenting the various 3-dimensional culture systems that have been applied for in vitro maturation of follicle:oocyte complexes. We also try to present an overview of outcomes with various biomaterials and animal models and also the limitations of the existing systems.

Techniques for cryopreservation of individual or small numbers of human spermatozoa: a systematic review

BACKGROUND Despite interest in cryopreservation of individual or small number of human spermatozoa, to date, little data is available as regards its effectiveness. We systematically reviewed the outcome after cryopreservation of individual or small numbers of human spermatozoa in patients with severe male factor of infertility. METHODS We searched the MEDLINE, EMBASE, Cochrane Systematic Reviews, CENTRAL, Web of Science, Scopus databases for relevant studies up to June of 2008. The search used terms referring to cryopreservation of small amount of sperm. Included studies were limited to human studies with no language restrictions. RESULTS We identified 30 reports including 9 carriers used for cryopreservation of small quantities/numbers of human spermatozoa (7 non-biological and 2 biological carriers). A wide variety of cryopreservation vehicles were reported. The recovery rate of spermatozoa cryopreserved in a known small number varied widely from 59 to 100%. Fertilization rates were in the range of 18-67%. Frozen-thawed spermatozoa, using this method, were subsequently used for intracytoplasmic sperm injection in only five studies, with few pregnancies reported so far. To date, there remains no consensus as to the ideal carrier for cryopreservation of small number of spermatozoa for clinical purposes. CONCLUSIONS Cryopreservation of individual or small numbers of human spermatozoa may replace the need for repeated surgical sperm retrieval. A controlled multicenter trial with sufficient follow-up would provide valid evidence of the potential benefit of this approach.

Single sperm cryopreservation on cryoloops: an alternative to hamster zona for freezing individual spermatozoa

Methodology enabling cryopreservation of individual spermatozoa in extreme cases of oligozoospermia would tremendously benefit patients. This study explores the use of a nylon loop for cryopreservation of small quantities of spermatozoa, and also describes a novel technique for freezing individual spermatozoa with this cryoloop. Experiments were conducted to compare sperm recovery and viability after cryopreservation in conventional vials versus on the cryoloops. Discarded human sperm specimens with varying parameters were utilized. The study also examines two different glycerolbased cryoprotectants, with and without test yolk buffer. For single sperm cryopreservation, 5-10 spermatozoa were selected and loaded onto cryoloops with the aid of a microscope and micromanipulation equipment. Sperm function testing was performed on both human and bovine spermatozoa frozen on cryoloops. Microquantities of spermatozoa frozen on cryoloops exhibited overall motility and viability parameters similar to control samples frozen in cryovials. Individually selected spermatozoa cryopreserved on loops were easily recovered and post-thaw motility was generally good. Sperm function testing demonstrated that both human and bovine spermatozoa cryopreserved on loops were able to undergo sperm head decondensation when injected into oocytes. Cryoloops may be an excellent alternative to hamster zonae for cryopreserving small numbers of human spermatozoa.

Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems

Human embryonic stem cells (hESC) have emerged as attractive candidates for cell-based therapies that are capable of restoring lost cell and tissue function. These unique cells are able to self-renew indefinitely and have the capacity to differentiate in to all three germ layers (ectoderm, endoderm and mesoderm). Harnessing the power of these pluripotent stem cells could potentially offer new therapeutic treatment options for a variety of medical conditions. Since the initial derivation of hESC lines in 1998, tremendous headway has been made in better understanding stem cell biology and culture requirements for maintenance of pluripotency. The approval of the first clinical trials of hESC cells for treatment of spinal cord injury and macular degeneration in 2010 marked the beginning of a new era in regenerative medicine. Yet it was clearly recognized that the clinical utility of hESC transplantation was still limited by several challenges. One of the most immediate issues has been the exposure of stem cells to animal pathogens, during hESC derivation and during in vitro propagation. Initial culture protocols used co-culture with inactivated mouse fibroblast feeder (MEF) or human feeder layers with fetal bovine serum or alternatively serum replacement proteins to support stem cell proliferation. Most hESC lines currently in use have been exposed to animal products, thus carrying the risk of xeno-transmitted infections and immune reaction. This mini review provides a historic perspective on human embryonic stem cell culture and the evolution of new culture models. We highlight the challenges and advances being made towards the development of xeno-free culture systems suitable for therapeutic applications.

The new Rapid-i carrier is an effective system for human embryo vitrification at both the blastocyst and cleavage stage

BackgroundThe Rapid-i is a new FDA cleared closed carrier for embryo vitrification. The cooling rate of - 1220°C/min is far lower than that reported with open vitrification systems such as the cryoloop (−15,000°C/min). Little published data is currently available on this device. This study presents our initial clinical data, as well as live birth outcomes, with the Rapid-i. The efficacy of this device for the cryopreservation of cleavage, as well as blastocyst stage human embryos is also analyzed. We further compare outcomes to those achieved with the cryoloop, an “open” vitrification system routinely used in our laboratory.MethodsHuman embryos were vitrified at either the 8–10 cell stage or else the blastocyst stage. The vitrification protocol was: 7.5% DMSO/7.5% ethylene glycol (EG) (2–3 min) followed by incubation in 15% DMSO /15% EG (45 sec) before loading on the vitrification carrier. Cryoprotectant was removed during warming by sequential washes in 0.25 M and 0.125 M sucrose in culture medium. Clinical outcome data for frozen cycles between January 2011 and August 2012 were stratified according to carrier and cell stage. The student t-test and chi square test were used to compare results. P value of < 0.05 was considered significant.ResultsA total of 486 vitrified-warmed embryos were assessed and 92% of them were transferred. The clinical pregnancy rate (CPR) and implantation rate (IR) with Rapid-i vitrified blastocysts were 59% and 49%, versus 47% and 37%, respectively for cleavage stage embryos. This was not statistically different from results with the cryoloop vitrified blastocysts (CPR 46%, IR 38%) nor the cleavage stage vitrified embryos (CPR 49%, IR 35%). To date, there have been 31 deliveries of 34 healthy infants from Rapid-i vitrified embryos, with another 12 pregnancies still on-going.ConclusionsThe Rapid-i offers an excellent alternative to existing open vitrification devices for embryo cryopreservation at the 8–10 cell stage as well as the blastocyst stage. Use of this type of “closed” sealed system that prevents direct contact between the embryos and liquid nitrogen reduces the potential risk of sample cross-contamination or infection. These preliminary data and live birth outcomes have paved the way toward transitioning to a closed vitrification system in our own IVF program.