Freezing Of Human Embryos Research Articles

A 3D Printed Vitrification Device for Storage in Cryopreservation Vials.

Sperm cryopreservation by vitrification is a promising approach for small-bodied animals such as zebrafish (Danio rerio). However, most vitrification tools adopted in aquatic research were initially designed for applications other than sperm (such as human embryo freezing) and, thus, pose challenges for adoption to sperm vitrification. Three-dimensional (3D) printing combined with open hardware sharing is an emerging strategy to address challenges in the development of cryopreservation tools. The goal of this study was to develop a 3D printed Vitrification Device for Cryo-Vials (VDCV) that can be integrated with the existing vial storage systems. The VDCV combined the vitrification and handling components to achieve functions of sample handling, vitrification, storage, and identification. The vitrification component featured a base, a stem, and a loop. A total of 36 configurations with various loop lengths (8, 10, and 12 mm); loop widths (2.0, 2.5, 3.0, and 3.5 mm); and support structures (open, transverse, and axial) of the VDCD prototypes were designed, fabricated, and tested. Device handling orientations (horizontal and vertical holding angles prior to and during freezing) were also investigated. Computer simulations estimated that the cooling rate of the samples ranged from 0.6-1.5 × 105 °C/min in all the configurations. Prior to freezing, loops with axial supports produced a minimum of 92% film retention. The overall trends of full vitrification occurrence were observed: horizontal plunging > vertical plunging, and axial support > transverse support and open loop. A loop length of 8 mm had the highest overall vitrification occurrence (86-100%). No significant differences (p = 0.6584) were shown in a volume capacity (5.7-6.0 μL) among the three supporting configurations. A single unit of VDCV can provide loading efficiencies of about 6 × 107 sperm/vial, pooling of samples from 3-6 males/vial, and fertilization for 1800 eggs/vial. The VDCV are low-cost (<$0.5 material cost per unit) and can be customized, standardized, securely labeled, and efficiently stored. The prototypes can be accessed by user communities through open-fabrication file sharing and fabricated with consumer-level 3D printers, thus facilitating community-level standardization.

Investigation of transfer results of human embryos that were vitrified and thawed at the cleavage, morula and blastocyst stages.

The objective of this study was to compare the rates of clinical pregnancy after the transfer of vitrified and thawed human embryos on days 3, 4 and 5 of embryonic development. In this retrospective study, the results of 148 embryo transfer cycles, using embryos frozen and thawed over the 3-year period between January 2016 and December 2018 at the Gülhane Training and Research Hospital Department of Gynecology and Obsterics Reproductive Medical Center of the University of Health Sciences, Ankara, Turkey were examined. Following embryo transfer - including 29 dissolved embryos frozen on day 3, 80 frozen on day 4, and 39 frozen on day 5 - results were examined in terms of clinical pregnancy rates. In this study, across all three groups, no significant differences were observed in terms of patient age, the number of oocytes collected, infertility reasons, the number of embryos dissolved, transfer day, or the number of embryos transferred. According to the transfer day, the rates of clinical pregnancy and ongoing pregnancy were significantly higher for embryos frozen on day 4 and transferred on day 5. Significantly higher rates of pregnancy and live birth were determined during in vitro fertilization (IVF) treatment with the freezing of human embryos on day 4 and the transfer of those embryos on day 5.

Controverses en AMP : faut-il congeler les ovocytes ou les embryons ? Les ovocytes

Since the beginning of IVF, cryopreservation concern spermatozoa or embryos due to the poor efficiency of oocyte freezing. To date, oocyte vitrification allows changing our practice privileging female gamete vitrification instead of human embryo freezing.

Live birth of healthy twins following transfer of re-cryopreserved blastocysts developed from frozen embryos recovered from a malfunctioning freezer

A computer-controlled cooling machine is the basic equipment required to perform slow freezing of human embryos. Although freezer malfunction is a rare event, it has been reported (HFEA press release. [1]). To date, there are no published reports regarding the fate of cryopreserved embryos encountered freezer malfunction during the slow cooling process. This case report describes our experience with a sudden temperature increase occurring during the slow cooling of human embryo cryopreservation. We successfully recovered cryopreserved embryos from the malfunctioning freezer by rapid thawing. Surviving embryos successfully developed to the blastocyst stage and were re-cryopreserved by vitrification. An ongoing twin pregnancy was achieved with two healthy twins following the transfer of two vitrified blastocysts.

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.

Application of intra- and extracellular sugars and dimethylsulfoxide to human oocyte cryopreservation

Oocyte cryopreservation may avoid many complications of human embryo freezing and provide future fertility for women undergoing cancer therapy. The objective of this study was to explore the application of intra- and extracellular sugars in combination with small amounts of dimethylsulfoxide (DMSO) to human oocyte cryopreservation as an alternative approach. Discarded human oocytes that were obtained from IVF patients under informed consent and IRB approval, were cryopreserved by slow cooling to -196 degrees C after being randomly distributed into three groups: (i) DMSO control without intra- and extracellular sugar; (ii) extracellular sugar (raffinose) + DMSO; and (iii) intra- and extracellular sugar (trehalose and raffinose, respectively) + DMSO. Subsequently, all cryopreserved oocytes were thawed rapidly, and their survival was assessed by morphological criteria after 24 h of culture. A total of 71 oocytes were evaluated in three groups with survival rates of 88.5% (23/26), 68.2% (15/22), and 52.2% (12/23) for intra- and extracellular sugar+DMSO, extracellular sugar+DMSO, and DMSO control groups, respectively. These results support the use of intra- and extracellular sugars as an alternative approach for cryopreservation of human oocytes.

Survival of Human Sperm Following Controlled- and Noncontrolled-Rate Cryopreservation/Die Überlebensrate menschlicher Spermatozoen nach einer kontrollierten und nicht kontrollierten Kryokonservierung

The purpose of this study was to develop a noncontrolled-rate (NCR) method of freezing semen in cryovials which could be used as a backup to controlled-rate (CR) freezing when the CR freezer was being utilized for freezing of human embryos or oocytes. Semen was frozen with three concentrations of glycerol (0%, 3.75%, 7.5%) in cryoprotective media in a CR freezer or using a newly developed NCR method of freezing. Semen was thawed in 40 degrees C water or in air at room temperature (RT). Controlled-rate freezing and 40 degrees C thawing resulted in significantly greater postthaw sperm motility and motility index compared with NCR freezing and RT thawing. Sperm velocity and linearity were not significantly effected by rate of freezing or thawing except when there was no glycerol present in the cryoprotective media. Postthaw motilities following CR-40 degrees C, NCR-40 degrees C, CR-RT and NCR-RT freezing-thawing were 25.6%, 24.8%, 21.6% and 18.3%, respectively. Because NCR freezing resulted in only a 0.8% decline in motility compared with CR freezing when semen was thawed at 40 degrees C it appears that this new NCR method of freezing can be successfully used as a backup to CR freezing when the CR freezer is being used for embryo or oocyte cryopreservation.

Freezing of cleavage stage human embryos negatively impacts metabolism compared to cryopreservation by vitrification

Slow freezing of human embryos conceived through IVF has been widely adopted for over two decades. However, in animal models slow freezing induces significant cellular trauma, including altered metabolism, which can compromise subsequent embryo development. In contrast, the process of ultra rapid vitrification has been shown to impart minimal cellular stress on gametes and embryos, and has now replaced slow freezing as the preferred method of cryopreservation in animal models. The aim of this work was to determine whether slow freezing and vitrification had different effects on human cleavage stage embryo metabolism and development. Laboratory study. Day 3 human embryos were donated for research with consent. Cleavage stage embryos underwent cryopreservation by slow freezing using 1,2-propanediol as the cryoprotectant. Alternatively, embryos underwent ultra rapid vitrification using the Cryoloop using ethylene glycol and 1, 2-propanediol as the cryoprotectants. Upon thawing or warming, embryos were incubated in 1 μl of G2 for 3h for metabolic analysis, and then cultured individually in 10 μl of G2 for 48h. Development to blastocyst and blastocyst quality were scored. The uptake of pyruvate on day 3 following slow freezing was 11.4 ± 0.9 pmol/embryo/h, compared to 19.1 ± 1.1 for embryos that had been vitrified (P<0.01). Blastocyst development and quality were enhanced in the vitrified group. Following warming, cryosurvived embryos (93.5%) were cultured for 24 h in medium G2 and then transferred. A mean of 2.4 embryos were transferred in 7 patients, resulting in a pregnancy rate of 57%. Tabled 1Table.Slow freezingVitrificationCryosurvival (%)98/110 (89.0)103/109 (94.4)Embryos with 100% blastomere survival (%)50/98 (51.0)80/103 (77.6)∗P<0.01.Blastocyst formation (%)49/98 (50.0)62/103 (60.1)Blasts.≥ 3 AA (%)21/49 (42.8)32/62 (51.6)Hatching blastocyst (%)10/49 (20.4)19/62 (30.6)∗ P<0.01. Open table in a new tab This study has shown for the first time that human embryo metabolism is significantly depressed following slow freezing. In contrast, embryos that were vitrified had a metabolism activity almost twice that of the frozen embryos. Subsequent embryo development appeared to be elevated following vitrification. These data support the hypothesis that vitrification is associated with less cellular trauma than slow freezing and should be considered as the primary method of human embryo cryopreservation.

Citation classics in Fertility and Sterility, 1975–2004

Citation classics in Fertility and Sterility, 1975–2004

Successful Cryopreservation of Mouse Oocytes by Low Concentrations of Trehalose and Dimethylsulfoxide

Oocyte cryopreservation may avoid some complications of human embryo freezing and preserve future fertility of young cancer patients. However, oocyte cryopreservation is still an experimental procedure, and there is a need for alternative approaches. In nature, various organisms effectively use sugars such as trehalose to undergo anhydrobiosis and cryobiosis. By overcoming the permeability barrier of cell membranes, we have previously shown that when present inside and outside of the cell, trehalose, a non-reducing sugar, protects mammalian cells including human and mouse oocytes against freezing-associated stresses.

Small amounts of microinjected trehalose protect mouse and human oocytes during freeze-thaw.

Objective: Oocyte cryopreservation would avoid legal and ethical problems associated with human embryo freezing and provide future fertility for women who are anticipating loss of gonadal function. However, the success rate of oocyte cryopreservation is still low. In nature, a variety of organisms including arctic frogs, tardigrades, and brine shrimp survive extreme dehydration conditions such as freezing and drought by accumulating sugars. The objective of this study was to examine whether sugars can be used as intracellular cryoprotectant for the cryopreservation of human oocytes.

Application of Vitrification to Human Embryo Freezing

To solve the problem of multiple pregnancies during the in vitro fertilization (IVF) and embryo transfer procedure, excess embryos must be cryopreserved for embryo transfer in future. We applied the vitrification method to cryopreservation of human embryos. A total of 31 frozen-thawed embryo transfer cycles were analyzed at the Yamagata University Hospital, Yamagata, Japan. The patients were introduced to IVF treatment and had an excess of valuable embryos to be frozen after the transfer of three fresh embryos that did not result in establishing a pregnancy. Excess human 8- to 16-cell stage embryos were exposed to vitrification solution and then frozen in liquid nitrogen. The cryoprotectant was removed by washing the embryos in media containing different concentrations of cryoprotectant. Three days after LH surge and/or 2 days after ultrasonographic ovulation the embryos were transferred. The rate of poor quality embryos significantly increased and the rate of good quality embryos decreased after thawing the embryos frozen by the vitrification method. In menstrual cycles with good quality embryo transfer, a higher rate of pregnancies was established than in the cycles in which fair or poor quality embryos were the highest grade of embryos transferred into the uterus. In total, 5 pregnancies were established from 31 embryo tansfers; 4 pregnancies were in cycles associated with the transfer of good quality embryos, and 1 pregnancy was in a cycle in which the highest grade of embryo was fair. When compared with slow embryo freezing methods, vitrification has marked advantages for clinical application in terms of cost and time. Vitrification will be an alternative method for embryo freezing.

To clone or not to clone--a Jewish perspective.

Many new reproductive methods such as artificial insemination, in vitro fertilisation, freezing of human embryos, and surrogate motherhood were at first widely condemned but are now seen in Western society...

Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme.

Mouse oocytes and embryos were obtained following ovulation induction of (C57B16 x CBA) F1 animals. Zonae pellucidae were exposed to alpha-chymotrypsin in phosphate-buffered medium (PB1) supplemented with 3 mg/ml bovine serum albumin upon a heated stage (37 degrees C) and were observed constantly through an inverted microscope. The endpoint of the bioassay was the limits of the zona no longer being seen clearly at x 200 magnification, and the time taken for each zona to dissolve was recorded. A dose-dependent response in dissolution time was clearly seen, with 1% alpha-chymotrypsin being chosen as the routine working solution. Cryopreservation of 2-cell mouse embryos using propanediol did not cause zona hardening but induced a small and significant softening, as gauged by the time taken for zona dissolution (2181 +/- 167 versus 1864 +/- 82 s). Zona hardening was not suspected to occur after the freezing of human embryos as there was no difference in implantation rates per embryo for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatment cycles between fresh [IVF: 63/644 (9.7%); ICSI: 51/330 (15.5%)] and frozen embryos [IVF: 36/458 (7.9%); ICSI: 18/112 (16.1%)]. Conversely, significant hardening of the zonae of mature oocytes was seen following cryopreservation (747 +/- 393 s) compared with freshly ovulated oocytes (151 +/- 68 s). It is concluded that (i) the freezing of murine oocytes with propanediol results in zona hardening, implying a possible benefit of ICSI after the cryopreservation of human oocytes, and (ii) the cryopreservation of embryos is not associated with zona hardening or reduced implantation, making microdissection of the zona in such cases generally unwarranted.

Pregnancies after transfer of ultrarapidly frozen human embryos.

We report our experience of freezing human embryos using an ultrarapid freezing method. The patients were superovulated. Oocytes were inseminated and cultured in HTF + 10% serum. A maximum of three embryos was transferred and the rest of the embryos were frozen ultrarapidly after a 3-min equilibration period in PB1 + 3.5 M DMSO + 0.25 M sucrose. Embryos were thawed in a 37 degrees C water bath for 6 sec, then cultured in PB1 + 20% serum for 10 min. The surviving embryos were transferred into patients on the same day of thawing. Sixty-three embryos were thawed, of which 52 embryos (83%) survived with at least one intact blastomere. Nineteen frozen-thawed embryo transfers were made. The mean embryos per transfer was 2.7. Three pregnancies (16%/transfer) were established. One miscarriage occurred in the eighth week of pregnancy. Two pregnancies went to term and three healthy infants were born. The present data demonstrate that ultrarapid freezing is a method worth consideration in the area of human embryo freezing.

Survival and pregnancy outcome after ultrarapid freezing of human embryos

Contrary to the belief that rapid cooling and thawing of mammalian embryos is detrimental to survival, it has been shown that under certain conditions mammalian embryos can survive rapid freezing-thawing. In this study, 237 human fertilized oocytes (93 pronucleates, 20 multipronucleates, and 124 cleaved embryos) were frozen for a period of 1 day to 7 months using the ultrarapid freezing system. After thawing, 94% of the embryos in the pronucleate group and 89.5% in the multipronucleate group showed normal morphological features, and 79% and 71%, respectively, started to cleave. Forty cleaved embryos were also frozen-thawed, but only 15 (37.5%) survived at thawing. Thirty-four frozen-thawed cleaved embryos were transferred to 20 patients during spontaneous cycles. Four patients became pregnant.

Survival and pregnancy outcome after ultrarapid freezing of human embryos

Contrary to the belief that rapid cooling and thawing of mammalian embryos is detrimental to survival, it has been shown that under certain conditions mammalian embryos can survive rapid freezing-thawing. In this study, 237 human fertilized oocytes (93 pronucleates, 20 multipronucleates, and 124 cleaved embryos) were frozen for a period of 1 day to 7 months using the ultrarapid freezing system. After thawing, 94% of the embryos in the pronucleate group and 89.5% in the multipronucleate group showed normal morphological features, and 79% and 71%, respectively, started to cleave. Forty cleaved embryos were also frozen-thawed, but only 15 (37.5%) survived at thawing. Thirty-four frozen-thawed cleaved embryos were transferred to 20 patients during spontaneous cycles. Four patients became pregnant.

Experimental cryopreservation: A model for the freezing of human embryos and oocytes

Experimental cryopreservation: A model for the freezing of human embryos and oocytes

Factors involved in the success of human embryo freezing. Does cryopreservation really improve the IVF results?

Factors involved in the success of human embryo freezing. Does cryopreservation really improve the IVF results?