{"paper_id":"72ca73d6-67a5-4bc0-90f6-b38954c23628","body_text":"Subsequent to  in vitro  maturation (IVM), as a novel \nexpanded technique, improved immature oocyte can \nbe used to treat infertile women. A variety of studies \nshowed successful fertilization, development of \nembryo and pregnancies with immature human oocytes \nmatured  in vitro  ( 1 ,  2 ). However, other studies in this \nfield revealed that the blastocyst performance of IVM \nis limited ( 3 ,  4 ).\nBefore implantation stage, embryo undergoes various \nprocesses including oocyte maturation, activation \nof embryonic genome and a transcription shift from \nmaternal to embryonic controls ( 5 ). Therefore, IVM \nprocess could be effective on the oocytes quality as any \ngrowth rate intervention would influence maturation \nof oocyte and further embryo development ( 6 ).\nDuring female period, maternal proteins/transcripts \ncontrol the embryonic developmental program until \noccurrence of embryonic genome activation (EGA). \nEGA is a process that occurs upon the first maternal \nmRNA degradation. After this stage, EGA occurrence \nwould happen at the human embryos in 4to 8-cell \nstage ( 7 ,  8 ). In one study, the gene expression profile \ncomparison in steps 5to 8-cell embryos, considering \nthe single blastomere level, was performed and \nrevealed novel EGA-related genes including  CCT3, \nDPPA5, MYC, POU5F1  and  CDH1 . One of the crucial \nstages of embryo development in 5to 8-cell stage is \nEGA, while the failure of this stage leads to embryo \narrest and final implantation failure ( 9 ).\nGenerally, morphological features of oocyte and\nembryo are common criteria for determining embryonic \ndevelopmental competence to select the greatest viability \n( 10 ,  11 ). A successful pregnancy establishment cannot\nrely on normal morphology of embryo. The comparison of\nbovine morulae and blastocysts between culture systems \nand protein supplements has revealed variations in the \ncomparative increase of some gene transcripts which are \ndevelopmentally important ( 12 ,  13 ).\nAccordingly, these gene expressions could be a \npotentially substantial marker for evaluating embryo \nviability and implantation ( 3 ). Several researches \nrevealed that there are significant variations in \nmRNA levels of some genes produced  in vitro  and\n in vivo  embryos ( 14 - 17 ). Currently, determination \nof mRNA abundance in bovine during primary stage \nof embryonic development serves as an appropriate \nquality marker for both  in vitro  and culture conditions \ntechniques ( 14 ,  18 ,  19 ). However, in the assisted \nreproductive treatment (ART) for clinical practice, it \nwould be difficult to apply these important markers \nat the present time. Major research is needed for the \ndevelopment of reliable markers to evaluate oocyte \nquality and embryo viability ( 3 ).\nGiven the above, this study attempted to identify \nrepresentative genes to characterize the EGA-related \ngenes in human embryos. Moreover, this research is \ngoing to answer whether these gene expressions are \ninfluenced by IVM culture during the growth phase \nof oocytes. We aimed to provide the first library of \nEGA-related gene expressions within human embryos, \nresulting from intracytoplasmic sperm injection into \noocytes matured  in vitro  compared to oocytes matured\n in vivo , to lay the foundations for subsequent studies.\n\nThis project was approved by the Ethics Committee \nof Shahid Beheshti University of Medical Sciences, \nTehran, Iran (SBMU.REC.1393.78). In this research, \nwritten and verbal informed consent was obtained from \nthe couples who were undergone ICSI/preimplantation \ngenetic diagnosis (PGD) conforming to standard \nprotocols. The research conducted from April 2014 \nto June 2016, including 19 couples who performed \nICSI-PGD cycles and 45 couples who underwent \nrepeated implantation failure (RIF) treatment. Two \ndifferent experiments were studied. We used immature \noocytes at the GV stage obtained from patients who \nunderwent RIF treatment ( in vitro  group). In addition, \nwe used normal biopsied embryos excess donated for \ndoing scientific research after embryo transfer ( in vivo  \ngroup). Considering the aim of study, data relevant to \nmaturation rate of immature oocytes at the GV stage \nwas evaluated through  in vitro  group. Fertilization \nrate (16-19 hours after sperm injection), cleavage rate \nand quality score of embryos (72 hours after sperm \ninjection) were examined in the two groups.\nIn this study, female factor infertility, including \nchronic anovulation, poly cystic ovarian syndrome \n(PCOS), endometriosis, low ovarian reserve (defined \nas five or fewer oocytes on retrieval) and male factor \ninfertility were the exclusion criteria. Female and \nmale patients were less than 35 and 45 years old, \nrespectively.\n\nA long protocol regimen was applied to control \novarian stimulation, consisting of gonadotrophin \nreleasing hormone (GnRH) agonist. Patients’ \nhypophyseal suppression was applied by 0.1 mg \nadministration of Decapeptyl (triptorelin, Ferring \nPharmaceutical, Germany) or Diphereline (triptorelin, \nIpsen Pharma, France) in the previous cycle midluteal \nphase. The GnRH analogue dose was decreased to 0.05 \nmg until the day of administering human chorionic \ngonadotrophin (hCG).\nFor ovarian stimulation of the patients, \nrecombinant follicle-stimulating hormone (FSH, \nGonal-F, MerckSerono, Germany), human \nmenopausal gonadotrophin (hMG, Menopur, \nFerring Pharmaceuticals) or high purified urinary \nFSH (Fostimon, IBSA, Switzerland) was used. \nTransvaginal ultrasound was used on the day 5 of \nstimulation to evaluate the effect of hormone therapy \non ovary. Subsequently, gonadotrophin doses were \nindividualized based on patients’ results. While two or \nmore follicles reached a mean diameter of 18-20 mm, \novulation was triggered with 10,000-IU HCG (IBSA). \nAfter 34-36 hours of hCG injection, oocyte retrieval \nwas carried out by transvaginal ultrasound guided \nfollicle aspiration.\n\nTo digest the mass of cumulus-oocyte complexes, \nhyaluronidase enzyme (Life Global, USA) was used \nafter oocyte retrieval. Metaphase II (MII) oocytes \nwith first polar body were used for intracytoplasmic \nsperm injection (ICSI) ( in vivo  group). In couples\nwith RIF treatment, GV oocytes ( in vitro  group) were\ncollected. The oocytes were incubated in 6% CO 2  and \n37°C (Memmert, Germany) under mineral oil (Irvine \nScientific, USA) for approximately one hour. They \nwere subsequently denuded with 1% hyaluronidase \nenzyme (Life Global, USA) and a hand-drawn glass \npipette.\nFor GV oocytes detection ( in vitro  group), an \ninvert microscope (SM2800, Nikon) with 200 fold \nmagnification was used. Our strategy for selecting \nGV oocytes was prominent nucleus in the cytoplasm \nhomogeneous and the oocytes without any defects in \nthe overall appearance. The GV oocytes were placed in \na 20-30 µl drop of a commercial IVM medium (oocyte \nmaturation medium, Sage Media, USA) supplemented \nwith 75 IU/ml FSH and 75 IU/ml luteinizing hormone \n(LH) according to the manufacturer’s instructions for \n24-30 hours. Oocytes with the first polar body could \nsubsequently be injected by ICSI.\n\nAfter 2-3 days of abstinence, semen samples were \ncollected via masturbation into a non-toxic sterile \ncontainer. After the liquefaction of the semen at 37°C, \n5% CO 2  in air for 30 minutes, we processed the samples \nby swim-up technique.\nOne milliliter of semen sample was placed in a sterile \nconical centrifuge tube and a 1.2 ml layer of the medium \n(Ham’s F-10, Sigma, USA) was poured gently over it. \nThe sample was centrifuged at 300-500 g for 5 minutes \nand the supernatant was discarded. The pellet was \ndiluted with 0.5 ml of medium. The tube was inclined \nat an angle of about 45° and was incubated for 1 hour \nat 37°C. After the swim-up technique, two aliquots \n(200 ml) of the processed spermatozoa were taken, one \nof which was used for sperm parameters assay and the \nother was used for ICSI. According to World Health \nOrganization standard, sperm parameters, including \nsperm concentration, morphology and motility were \ndetermined as more than 15×10 6  spermatozoa per ml, \nmore than 4% and more than 32%, respectively ( 20 ).\n\nAfter injection, oocytes were incubated in 20 µl \ndroplets of global total medium (Life Global) under \nequilibrated mineral oil (in a humidified atmosphere, \n5% CO 2  at 37°C). After 16-18 hours, the oocytes were \ntested for pronuclei appearance (conforming to the \nconventional routine practice). Zygotes were cultured \nin global total medium (Life Global) and on day 3, \ntheir developmental stage was evaluated.\nAfter 16-19 hours of sperm injection, fertilization \nrate was determined as the resulting zygotes percentage \nby counting quantity of two pronucleus cells from \nthe injected MII oocytes total number. Embryos \nwere scored for their quality 72 hours after ICSI. \nMorphology of the embryos was calculated according \nto previous research in this area ( 21 ). To determine the \nrate of cleavage, the cleaved fertilized embryos total \nnumber was computed on day 3 ( 22 ).\n\nOn day 3, post-ICSI, embryos were inserted in each\nmicrodrop of 5 µl of Ca 2+ /Mg 2+  free biopsy medium \n(LG PGD BIOPSY medium, Life Global) under \nmineral oil. After mechanical drilling of the zona \npellucida, one blastomere was gently removed by \na biopsy micropipette for analysis. The embryo was \nwashed two times in global total medium (Life Global) \nand transferred into a fresh medium drop for embryo\nvitrification. For 15 minutes at room temperature (2027.), \nthe embryo was maintained in an equilibration \nsolution (Kitazato BioPharma Co., Japan). Afterwards, \nthe embryos were aspirated for 1 minute period and then \ninserted into the vitrification solution (VS, Kitazato \nBioPharma Co.) at room temperature. Subsequently,\nthe embryos were inserted on cryotop with a minimum\nvolume of VS solution, and quickly plunged into liquid \nnitrogen ( 23 ).\n\nIn this research, genes that are either known as \ndevelopmental and pluripotency or used in other species \nas markers, were selected for embryo viability ( 14 ,  18 , \n 24 ), while they are involved in early embryogenesis \n( POU5F1, CDH1, MYC  and  DPPA5 ). The samples \nwere applied for RNA isolation, complementary \nDNA (cDNA) synthesis and quantitative reverse \ntranscriptase-polymerase chain reaction (qRT-PCR) \nanalysis. The extraction of total RNA from samples, \ncDNA synthesis, and qRT-PCR analysis were carried \nout according to the protocol explained in the previous \nstudies ( 25 ,  26 ).\nIn summary, sample was pipetted into the eppendorf \ntube containing 1.5 µl of lysis buffers. cDNA was \nsynthesized by adding 2 µl poly-N and 5 µl nuclease \nfree water to each 2 µl embryo sample. The samples \nwere inserted in a BioRad thermocycler for 5 minutes \nat 75°C for the performance of reaction. By performing \nreverse transcription (RT), the tubes were placed on \nice. 200 u RT enzyme (1 µl), 5× RT buffer (5 µl), 10 \nmM dNTP (3 µl), and 10 u RNase inhibitor (0.25 µl, all \nfrom Sigma) were added to the reaction. RT reaction \nwas performed at 25°C for 10 minutes, 37°C for 15 \nminutes, 42°C for 45 minutes and 72°C for 10 minutes. \nThen, the samples were kept at 4°C overnight.\nPrimer sequences were used for qRT-PCR, to study \nthe expression levels of  POU5F (OCT4), CDH1, MYC  \nand  DPPA5  using Rotor Gene Q instrument (Qiagen, \nUSA) ( Table 1 ). According to the DNA Master SYBR \nGreen I mix manuals (Roche Applied Sciences, USA), we \nperformed RT-PCR reactions in a total volume of 13 µl \nusing 1 µM of each specific primer for the individual genes \nand 1 µM synthesized cDNA. The reaction conditions \nwere 5 second at 95°C and 49 extension cycles of \nincubating 3 minutes at 95°C for denaturation, 15 seconds \nat 60°C, 10 seconds at 72°C for amplification. Melting \ncurve analysis was applied to approve the single gene-\nspecific peak of all amplification reactions.  Beta2M  \nwas applied as an endogenous reference gene for \n POU5F1, CDH1, MYC  and  DPPA5  to normalize the \nqRT-PCR. The authors carried out three replications \nand normalized fold-changes at each sample to that of \nendogenous internal mRNA levels ( 25 ,  26 ).\nDetails of primers used in quantitative real time polymerase chain reaction\n\nAll statistical analyses were performed using the \nStatistical Package for Social Sciences software, version \n22 (SPSS, USA). The means of embryo quality score, \ncleavage, and fertilization were measured using the nonparametric \nanalysis test (Mann-Whitney U-test). REST \n2009 software (Qiagen) was used to analyze relative \nlevels of gene expression for various genes of embryos \nfrom two groups. The data are expressed as percentage \nmeans ± SEM. P<0.05 was considered as statistically \nsignificant.\n\nGerminal vesicle oocytes were collected from the \ncouples who were treated (out of 45 couples). 3-6 \ngerminal vesicle oocytes were collected from each patient \non average ( Table 2 ). The data of 19 couples undergoing \nICSI-PGD cycles were used ( Table 3 ).\nIn a total number of 217 germinal vesicle oocytes, 144 \n(63.18%) reached to the stage of metaphase MII and 19 \n(8.83%) reached and arrested in the stage of metaphase \nMI. In the germinal vesicle, 54 (27.09%) oocytes were \narrested.\nThe maturation of oocytes after 24-30 hours culture\nDemographic characteristics of all evaluated intracytoplasmic sperm injection (ICSI) cycles\nThere was no significant variation between two groups,\nregarding the injected oocytes number. The embryo\nquality score, cleavage and fertilization rate of the two \ngroups are described in Table 3. In both groups, good \nquality (A-B) of embryos was used. Those embryos\nwith normal morphology were chosen for molecular\ninvestigations. The rate of fertilization was significantly \ndifferent between  in vitro  group (54.71% ± 2.08) and\n in vivo  group (83.84 % ± 2.80, P=0.003). The cleavage\nrate was different among these groups (40.33 % ± 3.82 \nvs. 79.61 % ± 221, P=0.0001). Embryo quality score \nwas decreased  in vitro  compared to  in vivo . However, no \nstatistically significant difference was observed in embryo \nquality score between  in vitro  (2.54 ± 0.67) and  in vivo \n(2.56 ± 0.3, P=0.733). Rates of 4-cell embryo formation \non day 2 of embryogenesis was significantly different \nbetween  in vitro  (72.12% ± 1.63) and  in vivo  (95.34 % ± \n2.65, P=0.005). Besides, rates of 8-cell embryo formation \non day 3 of embryogenesis was significantly different \nbetween  in vitro  (70.69% ± 1.39) and  in vivo  (88.64 % ± \n1.72, P=0.038).\nPCR was performed in order to determine the quantitative \nmRNA expression profile of the implicated EGA-related \ngenes ( POU5F1, CDH1, MYC  and  DPPA5 ) in embryos \noriginated from intracytoplasmic sperm injection. By \ncomparing relative transcription level of  POU5F1,\nMYC  and  DPPA5  in embryos derived from ICSI, a significant \ndifference was identified in the levels of pluripotency and \ndevelopmental genes among  in vitro  and  in vivo  groups \n(all data were P=0.0001).  CDH1  gene expression was \nreduced  in vitro  compared to  in vivo , while no statistically \nsignificant difference was observed between these two \ngroups (P=0.341,  Fig .1 ).\nEGA related genes transcript relative quantification. Relative \nexpression of mRNA of  POU5F1, CDH1, MYC  and  DPPA5  in 8-cell stage \nof human embryos  in vitro  group and  in vivo  group showed that there \nwas a significant statistical difference (relative expression of mRNA of \n POU5F1, MYC  and  DPPA5 ) between the two groups. The mRNA levels of \nthe genes were analyzed with quantitative real time-PCR. The mRNA level \nof each sample was normalized to those of Beta2m mRNA levels. Data are \npresented as mean ± SEM. ***; P<0.001.\n\nIn this research, we firstly aimed to evaluate the \nmaturation rate in IVM-GV oocytes. We used immature \noocytes at the GV stages which could be obtained from \npatients who underwent RIF treatment ( in vitro  group). GV \noocyte culture for 24-30 hours provides more embryos for \npatients with infertility. Routinely, our criteria for selected \nGV oocytes were the presence of a prominent nucleus in \na homogenous cytoplasm with any type of defect in the \noocyte’s overall appearance.\nIn this manner, we evaluated fertilization and cleavage\nrate in IVM-GV oocytes with  in vivo  matured oocytes \ncollected in couples who underwent ICSI-PGD cycles. \nAfter embryo transfer, the normal biopsied embryos \nexcesses were donated for scientific research ( in vivo  \ngroup). We cultured GV oocytes for 24-30 hours based \non IVM studies ( 12 ). The maturation rate of GV was \ndetermined to be 63.18% in our study, which is almost in \nagreement with other studies ( 27 ,  28 ).\nHowever, other studies reported about 35-78% IVM \nrate for GV oocytes collected in stimulated cycles ( 29 , \n 30 ,  32 - 36 ). In this study, fertilization rates in GV-matured\nvs.  in vivo  MII oocytes were significantly different, and \nthis is in agreement with the report of Kim et al. ( 27 ) \ndemonstrating that rates of fertilization are significantly \ndifferent in the IVM and  in vivo  groups. In the present \nwork, the percentage means of cleaving embryos which \ncould reach to the 4-cell stage was significantly different\n in vitro , compared to the  in vivo . It was reported that \nembryos had decreased in quality, and 4-cell embryos \nwere significantly lower in the IVM group on the second \nday ( 28 ).\nIn another study, the rate of IVM cleaved embryos \napproaching to the four-cell step after 40 hours was \n84.5% and it was not considerably different from  in vivo  \ngroup ( 27 ). Some studies reported that oocyte maturation \nand fertilization rates have been 63 and 62% in PCO and \nPCOS cases (34 IVM cycles) respectively ( 29 ), while \nthese rates were 74.3 and 72.6% for maturation and \nfertilization in 63 regular cycles ( 30 ).\nThere are variations in the rate of oocyte maturation, \nfertilization and cleavage of IVM oocytes in different \nstudies. Several factors affect maturation of oocytes. \nTherefore, many differences are expected between these \nresults. However, it would be reasonable to indicate \nthat a crucial factor is related to the culture of immature \noocytes. Thus, nuclear and cytoplasmic maturation must \nbe considered together for immature oocytes maturation \n( 3 ). So, we can conclude that the IVM process could \nbe effective in the oocytes maturation, in any growth \nphase intervention, and it would affect the next embryo \ndevelopment. It should be noted that morphological \ncriteria and morphometric measures of oocytes in GV \nstage, as nuclear and cytoplasmic competence predictor, \nwere not reliable ( 31 ).\nTo assess and predict an ART program success, oocyte \nmorphological analysis, as an oocyte quality marker, is \napplied using phase contrast microscopy ( 32 ). Comparison \nof the maturation and fertilization rates in these two \ngroups showed that this simple and practical criterion \nis not trustable for the GV oocytes selection and more \nattention should be paid to the conditions of GV oocytes \nculture. Culture condition was greatly affected by oocyte \nmaturation  in vitro . Moreover, the percentage of oocytes \ndeveloping blastocyst stage is an appropriate indicator \nof suitable conditions for the next oocyte development \nstages as well as the next embryo. Nevertheless, \nevidences show that morphological assessment could \nnot always be a benchmark determinant for the fertilized \noocyte and competence development ( 33 ). Results of this \nstudy indicated that, in addition to certainty of the IVM\nculture medium maturity and appropriate morphology of\nthe oocytes, other factors in the medium of IVM culture \nand oocytes can be effective in the next steps of embryo \ndevelopment.\nOur second purpose was to compare gene expressions \nrelated to EGA in human embryos generated from \nimmature and mature oocytes (matured  in vivo  and in \nvitro, prior to exposure to sperm) recovered from women \nundergoing gonadotrophin treatment for ART. Pluripotent \nmaintenance is considered to be among the most important \nprocesses that can be altered during embryo culture. \nPluripotency is largely controlled by three genes:  Oct4 \n(Pou5f1), Nanog  and  Sox2  ( 24 ). We assessed the EGA-\nrelated genes, including  POU5F1, CDH1, MYC (c-MYC)  \nand  DPPA5  ( 9 ), in two groups. This is the first research \nexploring the function of EGA-related genes pattern in \nART (embryo derived from oocytes matured  in vitro ).\nIn the previous investigations, embryonic development\nwas inhibited before reaching to the morula stage\ndue to prohibiting transcription with a-amanitin ( 34 ).\nDevelopment, under suboptimal  in vitro  conditions, will\nnot go beyond the stage where embryonic genome is\nactivated ( 35 ). Therefore, we proposed that ART could \nprofoundly impress the EGA pattern in the primary stage \nof embryo development.\nPrevious studies showed while only about 50% of the \nzygotes are able to progress into blastocyst stage, about \n80% of  in vitro -matured and  in vitro -fertilized bovine \noocytes may reach high cleavage rates ( 36 - 39 ). This \nhighlights culture periods and conditions importance for \nthe production, viability and blastocysts quality. Several \nbovine studies showed that in addition to the role of the \noocyte quality, the period of post-fertilization culture \nconditions could have great influences on the gene \nexpression patterns responsible for the development of \nembryo ( 16 ).\nIn this study, related genes to EGA process were \nevaluated.  POU5F1  and  DPPA5  are known to not only \nbe involved in the maintenance of embryonic stem \n(ES) cell pluripotency, but also play a key role in the \nembryos development. It was reported that POU5F1 null \nhomozygous embryos are arrested by the implantation \ntime ( 40 ).\nMYC is a regulator gene coding a transcription factor.\nThe multifunctional protein encoded by this gene is a\nnuclear phosphoprotein, which has an important role in \nprogression of cell cycle and cellular transformation ( 41 ).\n DPPA5  is another gene playing role in the maintenance of \nES cell pluripotency. In this study, the  POU5F1, MYC  and  DPPA5  gene expressions showed a significant decrease\n in vitro  group in comparison with  in vivo  group embryos, \ndespite the normal morphology for these embryos). No \nstatistically significant difference was observed for\nembryo quality score  in vitro  group versus  in vivo .\n CDH1  gene expression was reduced  in vitro  compared \nto  in vivo , but no statistically significant difference was \nobserved between these two groups. This study showed \nthat IVM has a negative influence on the level of the \npluripotency as well as developmental and EGA-related \ngene expressions, in the primary stages of human embryos \ndevelopment. This decline in a critical developmental \ngene expression can negatively affect the subsequent \ndevelopment of embryos.\nVarious factors can be effective in gene expression and \nIVM process, decreasing the introduced genes expression \nthrough affecting important factors. Thus, perhaps the \nreason for the low rate of pregnancy and implantation in \nIVM process is the lack of critical developmental-related \ngenes during the IVM process, considering that typical \npregnancy rates with IVM were determined to be 30-35% \nper retrieval with 10-15% implantation rates ( 3 ).\nGiven the importance of these genes in embryonic \ndevelopment, the influence of IVM process on expression \nof such genes could not be ignored. RNAs and proteins \nare accumulated in the oocyte cytoplasm during oocytes \ngrowth phase, supporting the early phase of embryonic \ndevelopment before the activation of embryonic genome \n( 42 ,  43 ). During the growth of oocyte and folliculogenesis, \nespecially at the end of oocyte growth phase, the \nembryonic genome could find an opportunity to activate, \ndue to the accumulation and subsequently degradation \nof many maternal mRNA species ( 44 ). During zygotic \ngenome activation (ZGA), necessary amount of maternal \nfactors may play a significant role ( 45 ).\nFor synthesis and accumulation of maternal factors, \nthe process of oocytes maturation should be completed \nincluding nuclear and cytoplasmic maturations. If these \nagents activity change, the EGA process will be affected. \nThe quality of oocyte culture medium is one of the crucial \nfactors that can affect oocyte maturation and synthesis of \nmaternal factors during IVM processing ( 46 ). Therefore, \nmolecular markers play a key role in evaluating the \ntechnical quality of IVM through embryonic development \nstages and our gene transcription knowledge. However, \nfurther studies would be necessary to evaluate the exact \ncause of reduced level of the introduced gene expressions \nand the IVM process improvement, to minimize the \nnegative effects of IVM and enhance the embryo growth.\n\nIt is widely accepted that maternal instructions greatly\naffect the embryonic development primary stages,\nwhich are fully loaded into the oocyte in the form of\nmRNA and proteins. Eventually, this maternal program \ncontrols the ZGA. This study showed that IVM process \nhas a negative influence on the fertilization and cleavage \nrate as well as the pluripotency and developmental \ngene expression levels ( POU5F1, MYC  and  DPPA5 ) in \nhuman preimplantation embryos. It can be deduced that \nnormal embryo morphology cannot be a suitable scale for \nsuccessful development of embryo during preimplantation \nstages. Therefore, further studies would be necessary to \nexamine the molecular level and improve culture media \nfor IVM.","source_license":"CC-BY-4.0","license_restricted":false}