scholarly journals A PMMA-Based Microfluidic Device for Human Sperm Evaluation and Screening on Swimming Capability and Swimming Persistence

Micromachines ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 793
Author(s):  
Yimo Yan ◽  
Haoran Liu ◽  
Boxuan Zhang ◽  
Ran Liu
Keyword(s):  
Human Sperm ◽  
High Viscosity ◽  
Control Group ◽  
High Quality ◽  
Vitro Fertilization ◽  
Step Procedure ◽  
Motion Characteristics ◽  
Selection Of

The selection of high-quality sperm is essential to the success of in vitro fertilization (IVF). As human cervical mucus has a high viscosity, without enough swimming persistence, human sperm clouds cannot arrive at the ampulla to fertilize the egg. In this study, we used swimming capability and motion characteristics that are known to be associated with fertilization ability to evaluate the quality of sperm. Here, a clinically applicable polymethyl methacrylate (PMMA)-based microdevice was designed and fabricated for sperm evaluation and screening for swimming capability and persistence in a viscous environment. In this study, we applied methylcellulose (MC) to mimic the natural properties of mucus in vivo to achieve the selection of motile sperm. Sperm motion was recorded by an inverted microscope. The statistical features were extracted and analyzed. Hundreds of sperm in two treated groups with different concentrations of MC and one control group with human tubal fluid (HTF) media were video recorded. This device can achieve a one-step procedure of high-quality sperm selection and achieve the quantitative evaluation of sperm swimming capability and persistence. Sperm with good swimming capability and persistence may be more suitable for fertilization in a viscous environment. This microdevice and methods could be used to guide the evaluation of sperm motility and screening in the future.

scholarly journals Piglets produced by transfer of embryos obtained by in vitro fertilization of oocytes matured in vitro with thymosin: A case report

2020 ◽  
Vol 76 (03) ◽  
pp. 6356-2020 ◽  
Author(s):  
KATARZYNA PONIEDZIAŁEK-KEMPNY ◽  
BARBARA GAJDA ◽  
IWONA RAJSKA ◽  
LECHOSŁAW GAJDA ◽  
ZDZISŁAW SMORĄG
Keyword(s):  
Case Report ◽  
In Vitro Fertilization ◽  
Control Group ◽  
First Birth ◽  
Vitro Fertilization ◽  
Research Material ◽  
Preliminary Study ◽  
Experimental Group

The aim of the study was to examine the in vivo viability of in vitro-produced (IVP) porcine embryos obtained from oocytes matured with thymosin. The research material for this study consisted of immature pig oocytes obtained from ovaries after slaughter and ejaculated semen obtained from one boar. The immature oocytes were cultured in vitro until the metaphase II stage in a medium supplemented with thymosin (TMS). The presumptive zygotes obtained were cultured in vitro for 4-40 hours. The presumptive zygotes and 2-4-cell embryos were evaluated in vivo after transferring them to synchronized recipients. After the transfer of embryos from the experimental group into 2 recipients (50 embryos into each gilt) and the transfer of 50 embryos from the control group into 1 recipient, both gilts that had received embryos obtained by in vitro fertilization of oocytes matured with TMS became pregnant and delivered a total of 16 live piglets. After the transfer of embryos from the control group, no pregnancy was achieved. In conclusion, the results of our preliminary study suggest that the maturation of pig oocytes with thymosin supports the in vivo survival of in vitro produced embryos. It is important to note, that this was the first birth of piglets obtained after transfer of IVP embryos in Poland.

186 THE EFFECT OF MATRIGEL ON THE DEVELOPMENT OF IN VITRO-FERTILIZED PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 209
Author(s):  
S.-W. Kim ◽  
M.-J. Lee ◽  
B.-C. Yang ◽  
G.-S. Im ◽  
H.-H. Seong ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Statistical Significance ◽  
Control Group ◽  
Fertilization In Vitro ◽  
Mouse Tumor ◽  
Matrix Product ◽  
Vitro Fertilization ◽  
Culture Systems

The application of matrix proteins to culture systems for growth of embryos is a logical extension in the quest to better simulate the in vivo culture environment. Matrigel, a commercially available extracellular matrix product containing collagen IV, laminin, entactin, and proteoglycans isolated from mouse tumor cells, has been tested. Development of mouse pre-implantation embryos cultivated in conventional culture medium was contrasted to that of embryos grown in solubilized Matrigel medium. In the solubilized Matrigel medium, in vitro blastocyst formation and hatching were significantly enhanced over that observed in the medium alone control. Therefore, the aim of this study was to investigate the effect of solubilized Matrigel on the development of porcine embryos after in vitro fertilization. In vitro-matured oocytes were fertilized in mTBM medium with fresh spermatozoa for 6 h. Putative zygotes were cultured in PZM-3 medium supplemented with (matrigel group) or without (control group) 0.8% Matrigel for 6 days. The number of cells in blastocysts was determined by staining with Hoechst 33342. Assessment of apoptosis in blastocysts was examined by TUNEL. The statistical significance of the data was analyzed using chi-square test and Student's t-test. The addition of Matrigel appeared not to increase the proportion of blastocysts (control: 71/219, 21.8 � 2.2% vs. Matrigel: 69/220, 23.5 � 5.8%). However, the mean cell numbers were significantly increased by Matrigel (Matrigel: n = 31, 52.9 � 18.1 vs. control: n = 30, 42.3 � 14.4; P < 0.01). The proportion of apoptotic cells was significantly lower in the Matrigel group (Matrigel: 4.5 � 4.2% vs. control: 6.6 � 5.5%; P < 0.05). In this experiment, Matrigel appeared to increase blastocyst quality of porcine embryos. Results suggest that Matrigel, as an extracellular matrix component, may be another avenue for formulating more physiological culture systems.

200 SYNCHRONIZATION OF OOCYTE MEIOTIC MATURATION IN SUPEROVULATED MICE IMPROVES IN VITRO FERTILIZATION RATE

2012 ◽  
Vol 24 (1) ◽  
pp. 212
Author(s):  
A. M. Taiyeb Ridha ◽  
D. C. Kraemer
Keyword(s):  
Embryonic Development ◽  
Fertilization Rate ◽  
Early Embryonic Development ◽  
Meiotic Maturation ◽  
Control Group ◽  
Vitro Fertilization ◽  
Development Rates ◽  
Oocyte Meiotic Maturation

In vitro synchronization of oocyte nuclear and cytoplasmic maturation has been found to improve the IVF rate of ovarian oocytes in several species, including humans, in comparison with nonsynchronized in vitro-matured oocytes. Here, we tested the hypothesis that synchronization of oocyte meiotic maturation by an in vivo system in superovulated mice would increase the oocyte fertilization rate when compared to that of conventional superovulated oocytes. Recently, we observed that cilostazol (CZL), a PDE3-I, was able to inhibit mouse oocyte meiotic maturation in both in vitro and in vivo systems. Administering CZL at 7.5 mg, 4 or 7 h pre-hCG allowed retrieval of ovulated oocytes of which >95% were at MI stage, scored by Nikon stereo microscope (SMZ 1500). A conventional superovulation program was adapted in all treated and their control groups, in which mice were injected with eCG and after 48 h with hCG (7.5 IU for each hormone). On the second morning, 13 to 14 h post-hCG, mice were killed and oocytes were collected from oviducts and in vitro fertilized (control). For the treated groups, CZL was administered in a single 7.5 mg oral dose (gavage) 4 or 7 h before the hCG injection. On the second morning, CZL-treated animals were killed at the same timing as control animals and oocytes were retrieved from the oviduct and in vitro matured for 6 h (for those gavaged with CZL, 4 h pre-hCG) or 3 h (for those gavaged with CZL, 7 h pre-hCG) to MII oocytes before IVF. These groups were designated as in vivo-in vitro synchronized/matured oocytes. In other groups treated with CZL, 4 or 7 h pre-hCG, the ovulated oocytes were allowed to mature in the oviduct (full in vivo synchronization and maturation) and oocytes were retrieved and fertilized with the same fertilization timings as the in vivo-in vitro synchronized/matured oocytes. Oocytes were cultured for 1 day after IVF and examined for cleavage. Statistical differences were analyzed by cross-tabulated chi-square test. The full in vivo synchronization and maturation (for both CZL dose timings of 4 and 7 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [89% (n = 219) and 92.2% (n = 374) vs 81.8% (n = 198); P = 0.034 and P < 0.0001, respectively]. The in vivo-in vitro synchronized/matured oocytes (CZL dose timing at 7 h, but not 4 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [88.5% (n = 339) vs 83.4% (n = 458), respectively; P = 0.043]. However, the increase of the IVF rate of the oocytes from mice treated with CZL, 4 h pre-hCG, in the in vivo-in vitro synchronized/matured group was not significantly different from the control group [88.5% (n = 399) vs 83.4% (n = 458), respectively; P = 0.43]. It is concluded from the present study that synchronization of oocyte meiotic maturation by the in vivo and in vivo-in-vitro protocols can increase the IVF rate of oocytes in superovulated mice.

191 IN VITRO CULTURE DURING OOCYTE MATURATION AND FERTILIZATION INFLUENCES THE TRANSCRIPTOME PROFILES OF BOVINE BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 186
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
F. Rings ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Oocyte Maturation ◽  
Microarray Data ◽  
In Vitro Maturation ◽  
Culture Conditions ◽  
Control Group ◽  
Bovine Embryos ◽  
Vitro Fertilization

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitro fertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivo produced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivo produced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared with in vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.

47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)

2017 ◽  
Vol 29 (1) ◽  
pp. 131
Author(s):  
T. Fujikawa ◽  
C. Kubota ◽  
T. Ando ◽  
S. Imamura ◽  
M. Tokumaru ◽  
...  
Keyword(s):  
Survival Rate ◽  
Germ Cell ◽  
Embryo Transfer ◽  
Conception Rate ◽  
Control Group ◽  
Vitro Fertilization ◽  
Polyamino Acid ◽  
Significant Difference

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34 v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P = 0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48 h and hatched rate until 72 h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v. CPLL: 31 v. 50), and recipients were diagnosed for pregnancy ultrasonically 50 days after embryo transfer. Conception rate of CPLL group was higher than control group (50.0% v. 29.0%; P = 0.063). In both experiments, the significant differences between control group and CPLL group were determined by chi-squared test. The effectiveness of CPLL in cells and embryos has been reported; however, there is no report using CPLL in bovine germ cells. In this research, CPLL improved the conception rate of AI and ET, probably due to its low toxicity and protection of the cell membrane. These results suggest that CPLL is available as a new cryoprotective material for bovine sperm and embryo in slow freezing methods.

scholarly journals Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions

Animals ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 329 ◽  
Author(s):  
Martina Colombo ◽  
Maria Giorgia Morselli ◽  
Mariana Riboli Tavares ◽  
Maricy Apparicio ◽  
Gaia Cecilia Luvoni
Keyword(s):  
Developmental Stages ◽  
Culture Conditions ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
In Vitro Embryo Production ◽  
Physical And Chemical

Cryoinjuries severely affect the competence of vitrified oocytes (VOs) to develop into embryos after warming. The use of culture conditions that provide physical and chemical support and resemble the in vivo microenvironment in which oocytes develop, such as 3D scaffolds and coculture systems, might be useful to improve VOs outcomes. In this study, an enriched culture system of 3D barium alginate microcapsules was employed for the in vitro embryo production of domestic cat VOs. Cryotop vitrified-warmed oocytes were in vitro matured for 24 h in the 3D system with or without fresh cumulus-oocyte complexes (COCs) in coculture, whereas a control group of VOs was cultured in traditional 2D microdrops of medium. After in vitro fertilization, presumptive embryos were cultured in 3D or 2D systems according to the maturation conditions. Vitrified oocytes were able to mature and develop into embryos in 3D microcapsules (17.42 ± 11.83%) as well as in 2D microdrops (14.96 ± 8.80%), but the coculture with companion COCs in 3D resulted in similar proportions of VOs embryo development (18.39 ± 16.67%; p = 1.00), although COCs presence allowed for blastocyst formation (0.95 ± 2.52%). In conclusion, embryos until late developmental stages were obtained from cat VOs, and 3D microcapsules were comparable to 2D microdrops, but improvements in post-warming conditions are still needed.

134 TRANSCRIPTOME PROFILE OF BOVINE BLASTOCYSTS DERIVED FROM ALTERNATIVE IN VIVO AND IN VITRO CULTURE CONDITIONS AT SPECIFIC PHASES OF EARLY EMBRYONIC DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 179 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Vitro Fertilization

An understanding of gene expression patterns due to altered environmental conditions during different time points of the pre-implantation period would improve our knowledge on regulation of embryonic development and improve success of embryo culture. The aim of this study was to examine the effect of alternative in vivo and in vitro culture conditions at specific phases of early embryonic development on transcriptome profile of bovine blastocysts. Using nonsurgical endoscopic oviducal transfer technology, 5 different blastocyst groups were produced. The first 2 groups were matured in vitro and then either transferred after maturation or after in vitro fertilization to synchronized recipients. The other 3 groups were matured, fertilized and cultured in vitro until 4-cell, 16-cell and morula stage before transfer. Blastocysts from each group were collected by uterine flushing at Day 7 and pooled in groups of 10. Complete in vitro (IVP) and in vivo blastocysts were produced and used as controls. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group vs the in vivo control group to examine the transcriptome profile of blastocysts. Compared with the in vivo control group, clear dramatic shifts were found in the number of differentially expressed genes (DEG, fold change ≥2) at 2 different time points. The first shift occurred for blastocyst groups that were transferred after in vitro fertilization and before embryonic genome activation (EGA). The second shift occurred for blastocyst groups that were transferred after EGA, as well as for the IVP group. Ontological classification of DEG showed that the more time spent under in vitro conditions, the higher the percentage of DEG involved in cell death and apoptotic processes. Moreover, lipid metabolism was the most significant process affected in the blastocysts transferred after in vitro maturation and blastocysts transferred at 16-cell stage. Most DEG involved in this process were down-regulated. Pathway analysis revealed that signalling pathways were the dominant pathways in all groups except the group that was transferred after in vitro maturation. That group showed significant down-regulation for genes involved in retinoic acid receptors activation pathways. These results showed that fertilization and EGA were the most critical developmental stages influenced by in vitro culture conditions and subsequently affect blastocyst quality, as measured in terms of gene expression patterns. Moreover, we identified molecular mechanisms and pathways that were influenced by altered culture conditions. These findings will enable the examination of strategies for modifying in vitro culture conditions at critical stages that will allow more efficient production of developmentally competent blastocysts.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide

Zygote ◽  
2011 ◽  
Vol 19 (4) ◽  
pp. 331-337 ◽  
Author(s):  
Mariana Groke Marques ◽  
Anibal Ballarotti Nascimento ◽  
Renato Pereira da Costa Gerger ◽  
José Sergio de Arruda Gonçalves ◽  
Ana Rita de Sousa Coutinho ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Culture Media ◽  
Cell Number ◽  
Mature Stage ◽  
Control Group ◽  
High Quality ◽  
Vitro Fertilization ◽  
Parthenogenetic Activation ◽  
Effect Of Treatment

SummaryThis study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 μg/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX– blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

scholarly journals Viscous Cervical Environment-on-a-Chip for Selecting High-Quality Sperm from Human Semen

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1439
Author(s):  
Manhee Lee ◽  
Jin Woo Park ◽  
Dongwon Kim ◽  
Hyojeong Kwon ◽  
Min Jeong Cho ◽  
...  
Keyword(s):  
Diffusion Mechanism ◽  
Female Reproductive Tract ◽  
Convection Flow ◽  
Dna Integrity ◽  
Cervical Canal ◽  
High Quality ◽  
Sperm Sorting ◽  
Selection Of

When ejaculated sperm travels through the vagina to the uterus, mucus secreted by the cervical canal generally filters out sperm having low motility and poor morphology. To investigate this selection principle in vivo, we developed a microfluidic sperm-sorting chip with a viscous medium (polyvinylpyrrolidone: PVP) to imitate the biophysical environment mimic system of the human cervical canal. The material property of the PVP solution was tuned to the range of viscosities of cervical mucus using micro-viscometry. The selection of high-quality human sperm was experimentally evaluated in vitro and theoretically analyzed by the convection-diffusion mechanism. The convection flow is shown to be dominant at low viscosity of the medium used in the sperm-sorting chip when seeded with raw semen; hence, the raw semen containing sperm and debris convectively flow together with suppressed relative dispersions. Also, it was observed that the sperm selected via the chip not only had high motilities but also normal morphologies and high DNA integrity. Therefore, the biomimetic sperm-sorting chip with PVP medium is expected to improve male fertility by enabling the selection of high-quality sperm as well as uncovering pathways and regulatory mechanisms involved in sperm transport through the female reproductive tract for egg fertilization.

scholarly journals Methotrexate impaired in-vivo matured mouse oocyte quality and the possible mechanisms

2019 ◽  
Author(s):  
Ning Tian ◽  
Dan-yu Lv ◽  
Ji Yu ◽  
Wan-yun Ma
Keyword(s):  
Oocyte Quality ◽  
Mouse Oocyte ◽  
Control Group ◽  
Vitro Fertilization ◽  
Embryo Formation ◽  
Blastocyst Quality ◽  
Chromosome Alignment ◽  
Chromosome Separation

Abstract Background: Methotrexate (MTX) is an antifolate agent which is widely used in clinic for treating malignancies, rheumatoid arthritis and ectopic pregnancy. As reported, MTX has side effects on gastrointestinal system, nervous system and reproductive system, while its potential damages on oocyte quality are still unclear. It is known that oocyte quality is essential for healthy conception and the forthcoming embryo development. Thus, this work studied the effects of MTX on the oocyte quality. Results: We established MTX model mice by single treatment with 5 mg/Kg MTX. Both morphological and molecular biology studies were performed to assess the in-vivo matured oocytes quality and to analyze the related mechanisms. The in-vivo matured oocytes from MTX-treated mice had poor in-vitro fertilization ability, and the resulting embryo formation rates and blastocyst quality were lower than the control group. We found that the in-vivo matured MTX-treated mouse oocytes displayed abnormal transcript expressions for genes of key enzymes in the folate cycles. MTX increased the rate of abnormal chromosome alignment and affected the regulation of chromosome separation via disrupting the spindle morphology and reducing the mRNA expressions of MAD2 and Sgo1. MTX reduced the DNA methylation levels in the in-vivo matured oocytes, and further studies showed that MTX altered the expressions of DNMT1 and DNMT 3b, and may also affect the levels of the methyl donor and its metabolite. Conclusions: MTX impaired the in-vivo matured mouse oocyte quality by disturbing folate metabolism and affecting chromosome stability and methylation modification.

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