Composition of protein supplements used for human embryo culture

Purpose

To determine the composition of commercially available protein supplements for embryo culture media and test if differences in protein supplement composition are biologically relevant in a murine model.

Methods

Amino acid, organic acid, ion and metal content were determined for 6 protein supplements: recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS). To determine if differences in the composition of these supplements are biologically relevant, mouse one-cell embryos were collected and cultured for 120 hours in each protein supplement in Global media at 5 and 20 % oxygen in an EmbryoScope time-lapse incubator. The compositions of six protein supplements were analyzed for concentrations of 39 individual amino acids, organic acids, ions and elements. Blastocyst development and cell cycle timings were calculated at 96-hours of culture and the experiments were repeated in triplicate. Blastocyst gene expression was analyzed.

Results

Recombinant albumin had the fewest undefined components , the lowest concentration of elements detected, and resulted in high blastocyst development in both 5 and 20 % oxygen. Buminate, LGPS and SPS had high levels of transition metals whereas SSS had high concentrations of amino acids. Pre-compaction mouse embryo development was delayed relative to embryos in AlbIX for all supplements and blastocyst formation was reduced in Buminate, SPS and SSS.

Conclusions

The composition of protein supplements are variable, consisting of previously undescribed components. High concentrations of pro-oxidant transition metals were most notable. Blastocyst development was protein dependent and showed an interaction with oxygen concentration and pro-oxidant supplements.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-014-0349-2) contains supplementary material, which is available to authorized users.     

低质量人类胚胎体外发育至囊胚能力的研究

目的:探讨临床上D3丢弃的低质量胚胎培养至囊胚的发育潜能,为从低质量胚胎获得胚胎干细胞提供依据。方法:收集IVF/ICSI35例,184枚低质量胚胎(包括异常受精胚胎),用序贯法微滴培养,D3～D9连续观察囊胚形成情况。结果:(1)184枚低质量胚胎于D4～D6形成23枚囊胚,囊胚形成率12.5%;(2)胚胎卵裂球数越多,囊胚形成率越高,胚胎级别越低,囊胚形成率越低;(3)3PN胚胎囊胚形成率低于2PN囊胚形成率(14.8%vs15.9%),3PN来源的胚胎具有一定的发育潜能。结论:低质量胚胎具有发育至囊胚的潜能,有可能成为分离胚胎干细胞的材料来源。     

Effect of culture medium volume and embryo density on early mouse embryonic development: Tracking the development of the individual embryo

Purpose

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly.

Methods

(1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining.

Results

The blastocyst development was significantly higher (P < 0.05) when nine embryos were cultured in 50 μl of droplet of culture medium compared with other volumes. The blastocyst development was significantly reduced (P < 0.05) in single embryo culture compared to group embryo culture with or without the WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P < 0.05) in single embryo culture with the WOW system than without.

Conclusions

Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced embryonic development with single embryo culture cannot be ameliorated by the WOW system.     

Effect of growth factors in culture medium on the rate of mouse embryo development and viability in vitro

The development of mouse embryos from two cells to blastocysts in Whittingham's T6 medium containing the growth factor insulin or the serum replacement NuSerum was studied and compared with development in vivo. The rate of embryo development to blastocysts was increased by the addition of Nu-Serum to T6 and there was a significant increase in the implantation rate of embryos grown in T6 + Nu-Serum compared with T6 alone or with insulin. However, the rate of embryo development in vitro was retarded compared with that in vivo and the number of normal fetuses following transfer to recipients of blastocysts grown in T6 + Nu-Serum was not different from the number for those grown in T6 alone or with insulin. The addition of neither insulin nor Nu-Serum to T6 optimizes embryo development or viability.     

Effect of polyvinylpyrrolidone on sperm function and early embryonic development following intracytoplasmic sperm injection in human assisted reproduction

The objective here was to review the effects of polyvinylpyrrolidone (PVP) upon sperm function and embryonic development in humans. PVP has been used successfully in intracytoplasmic sperm injection (ICSI) to facilitate the handling and immobilization of sperm for both domestic animals and humans. In our previous reports, PVP solution exists locally in embryos injected during the early developmental period, and also exerts influence over the developmental capacity of such embryos. In other reports, PVP causes significant damage to sperm membranes that can be detected by transmission electron microscopy, and has been associated with chromosomal abnormalities in pregnancy derived from ICSI embryos. In some Japanese clinics, PVP-free media has been used for sperm immobilization in order to optimise safety. Consequently, it is strongly suggested that the success rate of fertilization and clinical pregnancy could be improved by using PVP-free solution for human ICSI. In conclusion, our interpretation of the available data is to perform ICSI without PVP or select a lower concentration of PVP solution in order to reduce safety for pregnancy and children born via ICSI.     

妊娠肝内胆汁淤积症孕妇胎儿总胆酸水平对胎儿胰腺内分泌功能及胎儿生长发育的影响

目的 探讨妊娠肝内胆汁淤积症(ICP)孕妇的胎儿总胆酸水平与胎儿胰腺内分泌功能变化的关系及其对胎儿生长发育的影响.方法 选择2007年3月至2008年2月在中南大学湘雅二医院妇产科行剖宫产分娩的30例单胎ICP孕妇为ICP组,同期行剖宫产分娩的30例正常单胎孕妇为对照组.采用放射免疫法测定两组新生儿脐动脉血中胰岛素、胰高糖素水平;循环酶法测定总胆酸水平;氧化酶-过氧化物法测定血糖水平.并测量两组新生儿出生体重、身长,计算肥胖指数(PI).结果 (1)ICP组新生儿脐动脉血中胰岛素水平为(9.0±3.3)mU/L、胰岛素/胰高糖素比值为0.048±0.028,分别低于对照组的(10.1±3.7)mU/L及0.050±0.020,差异有统计学意义(P<0.05);ICP组新生儿脐动脉血中总胆酸水平为(10.3±3.8)μmol/L、胰高糖素水平为(235±57)ns/L,分别高于对照组的(4.1±1.3)μmol/L及(205±34)ng/L,差异有统计学意义(P<0.05);ICP组新生儿脐动脉血中血糖水平为(3.4±1.1)mmol/L,对照组为(3.6±1.2)mmol/L,两组比较,差异无统计学意义(P>0.05).(2)ICP组新生儿出生体重及身长分别为(3163±478)g及(46.5±2.3)cm,对照组分别为(3498±393)g及(49.3±1.9)cm,两组分别比较,差异均有统计学意义(P<0.01);ICP组新生儿PI(3.13±0.23)明显高于对照组(2.92±0.29),差异有统计学意义(P<0.01).(3)ICP组新生儿总胆酸水平分别与胰岛素、胰高糖素水平及胰岛素/胰高糖素比值呈直线关系,且随着总胆酸水平的升高,胰岛素水平及胰岛素/胰高糖素比值均降低,胰高糖素水平升高(P<0.01);ICP组新生儿脐动脉血中胰岛素水平及胰岛素/胰高糖素比值分别与出生体重、身长呈正相关,与PI呈负相关(P均<0.01);而胰高糖素水平与出生体重、身长呈负相关,与PI呈正相关(P均<0.01).结论 ICP孕妇的胎儿存在胰岛素分泌不足,胰高糖素分泌增多,胰岛素/胰高糖素比值下降的情况,其变化与脐动脉血总胆酸水平密切相关;胎儿胰腺内分泌功能变化可能影响胎儿的生长发育.     

Measurement of uptake and incorporation of nucleic acid precursors by preimplantation mouse embryos after development in vivo and in vitro

Purpose To assay DNA and RNA synthesis by developing mouse embryos in vitro and in vivo, we measured the uptake and incorporation of³ H-thymidine and³ H-uridine by morulae and blastocysts. We also evaluated the effect of adding EDTA to the culture medium on the uptake and incorporation of nucleic acid precursors by blastocysts.Results Thymidine and uridine incorporation increased after morulae developed into early blastocysts both in vitro and in vivo. However, the rates of uptake and incorporation were significantly lower by embryos grown in vitro than by those grown in vivo. The ratios of incorporation to total uptake were similar in embryos grown in vitro and in vivo. EDTA (100 µM) added to the culture medium significantly increased the incorporation of uridine into RNA by blastocysts grown in vitro (P <0.01) but did not increase the total uptake of uridine. Conclusion These observations showed that both DNA and RNA synthesis increased during the early development of preimplantation embryos and that those activities were reduced in embryos undergoing development in vitro. The results also suggested that through the mechanism of EDTA effect in embryo culture remains unknown, it appeared to reduce the retardation of RNA synthesis by embryos cultured in vitro through a selective stimulation of uridine incorporation.     

新建体外受精实验室中挥发性有机化合物对鼠胚体外发育的影响

目的:探讨新建成的体外受精(IVF)胚胎实验室挥发性有机化合物(VOC)的浓度变化及不同浓度VOC环境下对小鼠IVF和胚胎发育的影响和去除实验室内VOC的方法。方法:按照胚胎在培养环境中是否接触培养室内空气分为实验组和对照组,在实验室装修后每个月测定两组中VOC水平,比较两组不同浓度的VOC对小鼠体外受精率、2-细胞率、卵裂率和囊胚率的影响,比较排风扇抽风、Coda空气净化器和单纯活性炭吸附去除室内VOC的效果。结果:1装修后第1个月,实验组VOC浓度高于对照组(384.00±5.16 ppb vs 199.00±2.58 ppb,P0.05);装修后第2个月,实验组VOC浓度高于对照组(279.50±3.70 ppb vs 201.75±3.50 ppb,P0.05);装修后第3个月两组VOC浓度比较,差异无统计学意义(P0.05)。2装修后第1、2个月时实验组受精率、2-细胞率、卵裂率和囊胚率低于对照组,差异均有统计学意义(P0.05);装修后第3个月时两组以上4项指标差异无统计学意义(P0.05)。3空气净化器的VOC去除率[(60.15±2.18)%]明显高于排风扇抽风[(51.88±2.23)%]和单纯活性炭吸附[(28.95±1.35)%],差异有统计学意义(P0.05)。结论:实验室内高浓度的VOC会对小鼠IVF胚胎造成严重的胚胎毒性,导致胚胎质量下降和发育受阻;空气净化器是一种有效地去除实验室内VOC的方法。     

Remodelling of the bovine placenta: Comprehensive morphological and histomorphological characterization at the late embryonic and early accelerated fetal growth stages

IntroductionPlacental function impacts growth and development with lifelong consequences for performance and health. We provide novel insights into placental development in bovine, an important agricultural species and biomedical model.MethodsConcepti with defined genetics and sex were recovered from nulliparous dams managed under standardized conditions to study placental gross morphological and histomorphological parameters at the late embryo (Day48) and early accelerated fetal growth (Day153) stages.ResultsPlacentome number increased 3-fold between Day48 and Day153. Placental barrier thickness was thinner, and volume of placental components, and surface areas and densities were higher at Day153 than Day48. We confirmed two placentome types, flat and convex. At Day48, there were more convex than flat placentomes, and convex placentomes had a lower proportion of maternal connective tissue (P < 0.01). However, this was reversed at Day153, where convex placentomes were lower in number and had greater volume of placental components (P < 0.01- P < 0.001) and greater surface area (P < 0.001) than flat placentomes. Importantly, embryo (r = 0.50) and fetal (r = 0.30) weight correlated with total number of convex but not flat placentomes.DiscussionExtensive remodelling of the placenta increases capacity for nutrient exchange to support rapidly increasing embryo-fetal weight from Day48 to Day153. The cellular composition of convex placentomes, and exclusive relationships between convex placentome number and embryo-fetal weight, provide strong evidence for these placentomes as drivers of prenatal growth. The difference in proportion of maternal connective tissue between placentome types at Day48 suggests that this tissue plays a role in determining placentome shape, further highlighting the importance of early placental development.     

Retinoic acid and fibroblast growth factor-2 play a key role on modulation of sex hormones and apoptosis in a mouse model of polycystic ovary syndrome induced by estradiol valerate

ObjectiveThe main goal of the present study is to investigate the effects of retinoic acid and fibroblast growth factor-2 on serum levels of FSH and LH, histology, and apoptosis in the mouse model of Poly Cystic Ovary Syndrome (PCOS).Materials and methods80 female NMRI mice have been randomly divided into eight groups. Group 1 received normal saline as a control, and Group 2 received estradiol valerate (EV) at 4 mg/100 g of body weight. Moreover, Groups 3–4 were administered with RA (a dose of 0.05 μg/μl) and FGF2 (a dose of 0.01 μg/kg), respectively. Groups 5 and 6 respectively received the EV plus the RA (0.05 μg/μl) and FGF2 (0.01 μg/kg). Group 7 received the RA and FGF2 at doses corresponding to healthy mice, and Group 8 received the EV plus the RA + FGF2 (similar to previous doses). RA and FGF2 were injected three times per week for four weeks. Finally, histological and immunohistological parameters of the ovary were evaluated.ResultsThe study revealed that both single and combined injection of fibroblast growth factor-2 (FGF2) and retinoic acid (RA) in groups 5, 6, and 8 significantly reduced follicular diameters compared to group 2. Measurements confirmed that simultaneous injection of RA and FGF2 into polycystic mice significantly increased antral follicles, corpus luteum (CL), epithelial thickness, and oocyte diameter as well as decreased cystic follicles. Positive TUNEL cells that were considerably increased in the antral follicle of group 2 significantly decreased in the RA and FGF2 recipient groups, either alone or in combination. Besides, the injection of FGF2 increased preantral follicles and CL.ConclusionThe findings of the present investigation reveal that injection of RA and FGF2 has both protective and ameliorative effects that can promise new therapies for women with PCOS.     

Effects of group culture on the development of discarded human embryos and the construction of human embryonic stem cell lines

Purpose

To explore the effect of group culture on the developmental potential of discarded embryos in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles and establish the human embryonic stem cell lines for future research.

Methords

Fresh discarded embryos were collected from the IVF/ICSI-ET program in the reproductive medical center of the first affiliated hospital of Zhengzhou university in this study. All zygotes were individually cultured from Day 1 to Day 3. On Day 3, discard embryos were then cultured in group of 1–4 embryos per droplet (30 μl/droplets) with a constant culture medium until Day 5 or 6. Mechanical method was used to isolate the inner cell mass (ICM) of blastocyst from the embryo. Then we inoculated the ICM on feeder layer. After identification of those cells, the human embryonic stem cell lines (hESCs) were established.

Results

In this study, we collected 1,223 fresh discarded embryos and they were sequential cultured to the blastocysts (18.07 %, 221/1,223), in which good quality blastocysts were 61(4.98 %, 61/1,223). There was no significant difference in the patients. The embryos from 1PN, 2PN, 3PN were sequential cultured to the blastocyst s(39.31 %,92/234;12.87 %,64/497;13.21 %,65/492),in which good quality blastocysts was 13.6 %(32/92),2.61 %(13/64), 3.04 %(15/65).1PN embryo’s blastulation rate and quality embryo formation rate was significantly higher than the 2PN and 3PN embryos’ (P <0.05). Three embryos group cultivation has the highest blastulation rate and quality embryo formation rate (P <0.05). In total, we successfully established 4 hESCs lines.

Conclusion

The group culture of human discard embryos can improve the blastulation rate and blastocyst quality to some extent. Three embryos group cultivate is the better culture number. Human discard embryos are good source for establishment of hESCs.     

胚胎发育速度及形态评级和受精卵原核评级联合选择移植胚胎的研究

目的探讨胚胎发育速度及形态评级并参考受精卵原核评级（联合评级）,对行体外受精-胚胎移植及卵母细胞质内单精子注射后移植胚胎筛选的意义.方法回顾性分析我院2003年5-12月,采用联合评级筛选进行胚胎移植的434个周期,共2714个正常受精卵的资料.根据受精卵原核发育是否同步,分为原核发育同步组和原核发育不同步组,观察不同原核等级受精卵的发育潜力;首先根据胚胎发育速度及形态评级,再根据受精卵原核等级的联合评级选择移植胚胎.根据移植胚胎中是否含有原核发育同步的胚胎,比较原核发育同步组与原核发育不同步组受精卵进行胚胎移植后的临床妊娠率和着床率.结果2714个正常受精卵中,原核发育同步组受精卵1774个,其中优质胚胎743个,优质胚胎率为41.88%;原核发育不同步组受精卵940个,其中优质胚胎319个,优质胚胎率为33.94%,两组比较,差异有统计学意义（P＜0.01）.原核发育同步组中胚胎移植周期395个,临床妊娠率为47.85%（189/395）,着床率为27.49%（273/993）;原核发育不同步组中胚胎移植周期39个,临床妊娠率为43.59%（17/39）,着床率为25.00%（21/84）.两组比较,差异无统计学意义（P＞0.05）.结论采用联合评级,受精卵原核发育同步组与发育不同步组胚胎的临床妊娠率及着床率无差异;参考原核评级不能预测更高的妊娠率和着床率,但能预测胚胎发育的潜力.     

Dexamethasone attenuates the embryotoxic effect of endometriotic peritoneal fluid in a murine model

Purpose

The in vitro fertilization (IVF) pregnancy rate of women with advanced stage endometriosis is nearly half that of the general population, suggesting incomplete targeting of the pathophysiology underlying endometriosis-associated infertility. Compelling evidence highlights inflammation as the etiologic link between endometriosis and infertility and a potential target for adjunctive treatment. The objective of this study was to examine the effect of dexamethasone on murine embryos exposed to human endometriotic peritoneal fluid (PF) using the established murine embryo assay model.

Methods

PF was obtained from women with and without severe endometriosis. Murine embryos were harvested and randomly allocated to five groups of culture media conditions: (1) human tubal fluid (HTF), (2) HTF and 10 % PF from women without endometriosis, (3) HTF and 10 % PF from women with endometriosis (PF-E), (4) HTF with PF-E and 0.01 mcg/mL dexamethasone, and (5) HTF with PF-E and 0.1 mcg/mL dexamethasone. Embryos were cultured in standard conditions and evaluated for blastocyst development.

Results

A total of 266 mouse embryos were cultured. Baseline blastulation rates were 63.6 %. The addition of peritoneal fluid from women with endometriosis decreased the blastocyst development rate to 38.9 % (P = 0.008). The addition of 0.1 mcg/mL of dexamethasone to the culture media restored the blastulation rate to near baseline levels (61.2 %; P = 0.019).

Conclusions

The results of our in vitro study demonstrate the capacity of dexamethasone to mitigate the deleterious impact of endometriotic PF on embryo development. If confirmed in vivo, dexamethasone may prove a useful adjunct for the treatment of endometriosis-associated infertility.     

The effect of two distinct levels of oxygen concentration on embryo development in a sibling oocyte study

Purpose

This prospective randomized study used sibling oocytes of 258 women with ≥8 oocytes to compare the effect of 5 % O₂ versus 20 % O₂ concentrations on embryo development and clinical outcome.

Methods

Oocytes of each case were divided between incubators with either 5 % or 20 % O₂ concentration. Outcome measures were fertilization, cleavage, embryo quality, blastocyst formation, and implantation, pregnancy and live birth rates.

Results

Fertilization and cleavage rates were similar in both groups. The 5 % O₂ group had significantly more blastomeres (P < 0.05) and more top-quality embryos on day 3 (P < 0.02), as well as significantly more available embryos for transfer (31.6 % vs. 23.1 % for the 20 % O₂ group; P < 0.0001). There were significantly more cycles with good embryos in the 5 % group (76/258) than in the 20 % group (38/258) (P < 0.0001). Implantation and pregnancy rates were significantly higher for 5 % O₂ embryos (P < 0.03 and P < 0.05, respectively). Live birth rates per embryo transfer were 34.2 % and 15.8 %, respectively, P < 0.05.

Conclusions

Implantation, pregnancy and live birth rates are higher, and more good quality embryos are available for transfer and freezing with reduced rather than with atmospheric oxygen concentrations during embryo incubation.     

Prognostic value of growth of 4-cell embryos on the day of transfer in fresh IVF-ET cycles

Purpose

To investigate the prognostic value of growth of 4-cell embryos on the day of transfer in determining clinical pregnancy and live birth rates after fresh in vitro fertilization (IVF)-embryo transfer (ET) cycles.

Methods

Retrospective cohort study of all patients between January 2008 and January 2013 initiating fresh IVF-ET cycles resulting in embryos that were not more than 4 cells 72 h after oocyte retrieval in the morning of their transfer. Patients were stratified into 2 groups based on whether embryos did or did not grow more than the 4-cell stage on the afternoon of ET. The odds of clinical pregnancy and live birth were considered as primary outcomes. Student’s t-tests and Chi-square (χ2) tests were used as inidicated, with logistic regression controlling for maternal age and number of embryos transferred.

Results

Three hundred forty three patients were identified for inclusion: 165 and 178 patients had 4-cell embryos with and without growth on the afternoon of ET, respectively. The demographic and baseline IVF cycle characteristics of the study cohort were comparable. Patients with embryo growth had higher clinical pregnancy (13.9 % vs. 4.49 %) and live birth (10.9 % vs. 3.37 %) rates compared to patients without embryo growth. This represented an overall increased odds of clinical pregnancy [Odds ratio (OR) = 3.44; 95 % Confidence Intervals (CI) 1.49–7.93; P = 0.004)] and live birth (OR = 3.51; 95 % CI 1.36–9.07; P = 0.01). The increased odds remained unchanged after adjusting for maternal age and number of embryos transferred.

Conclusions

Transfer of 4-cell embryos 3 days after oocyte retrieval can result in clinical pregnancies and live births, albeit at a low rate. Growth of an embryo more than the 4-cell stage on the afternoon of ET may serve as a positive prognostic factor for IVF-ET cycle outcome.     

Stimulatory and inhibitory effects of amino acids on the development of hamster eight-cell embryos in vitro

Amino acids were added to a simple medium (TALP) in an effort to enhance the development of hamster eightcell embryos in vitro. The addition of 1.0 mM glutamine to TALP medium markedly increased the percentage of embryos reaching the blastocyst stage. The addition of groups of 4 or 20 amino acids to TALP + glutamine medium produced differential effects, some groups inhibiting development while others stimulated it. The beneficial effect of glutamine was on the eight-cell to morula transition as well as on blastocyst formation, while the effects of the other 20 amino acids studied were only on blastocyst development. This study represents further progress made in identifying the culture requirements for growth of hamster preimplantation embryos in vitro.