Abstract Study question Does embryo compaction positively correlate with blastocyst development and further embryonic ploidy status? Summary answer Fully compacted embryos develop into good-quality blastocysts, have shorter developmental times, and are related to ploidy status. What is known already To succeed in assisted reproduction technique (ART), it is necessary to select embryos that have the highest potential. So, numerous studies make an effort to establish parameters for selecting embryos. A Time-lapse system (TLS) allows embryologists to understand dynamic embryo change through continuous monitoring. In development, embryos undergo dynamic functional changes during compaction, which play a crucial role in blastocyst formation. It is also known that embryonic genome activation can be seen with compaction. Incomplete compaction leads to blastocyst developmental failure. Nevertheless, the details about the compaction of human embryos have not been paid sufficient attention, so still rarely known. Study design, size, duration This was a retrospective cohort study including couples that underwent an IVF cycle at the CHA Fertility Center, Gangnam, between January 2019 to October 2022. A total of 371 reached the blastocyst from 113 patients cultured in the TLS were analyzed. Among these, 94 blastocysts were analyzed by preimplantation genetic testing for aneuploidy (PGT-A). Statistical analysis was performed by prism9 using a t-test and chi-square test. P values <0.05 were regarded as statistically significant. Participants/materials, setting, methods Embryos were classified into two categories by compaction pattern: fully compacted (Group1, N = 194) and partially compacted (Group2, N = 177). Blastocyst quality was determined by morphology and divided into three groups (Good, Average, and Poor). The developmental time ranging from morula to blastocyst was annotated based on the embryo scope image. The surface of the blastocyst was measured every hour starting at blastocyst formation (tB) by using the ellipse tool of the Embryo Viewer software. Main results and the role of chance Good and average quality blastocysts are significantly higher in Group 1 than in Group 2 (21.6% vs. 3.4%, p < 0.01; 47.9% vs. 26.6%, p < 0.01, respectively). In contrast, poor-quality blastocysts are lower in Group 1 than in Group 2 (30.4% vs. 70.1%, p < 0.01). The beginning and completion of compaction, and blastocyst formation times of embryos from Group 1 were significantly shorter than those of embryos that Group 2(78.6h vs. 82.4, p < 0.01; 87.0h vs. 92.2h, p < 0.01; 100.2h vs. 103.7, p < 0.01, respectively). Also, there is a significant difference in longitudinal surface area at 3, 6, and 12h from the time of tB between the two groups. Consequently, the average expansion rate in Group 1 was significantly faster than in Group 2 (653.6 μm2/hour vs. 499.2μm2/hour, p < 0.05). According to the result of PGT-A, Group 1 had statistically significantly higher euploid and lower aneuploidy rates compared to Group 2 (47.2% vs. 36.2%, p < 0.001; 52.8% vs. 63.8%, p < 0.001, respectively). However, in the PGT-A group, there was no significant difference in developmental time between the two groups, regardless of fully or partial compaction. Meanwhile, the average expansion rate in euploidy blastocyst was significantly faster than in aneuploidy blastocyst (747.8 μm2/hour vs. 564.3 μm2/hour, p < 0.05). Limitations, reasons for caution The main limitation is the single-center retrospective approach. Therefore future prospective research is needed to verify and extend our findings. Another one, in the PGT-A there was no difference in developmental time regardless of fully or partially compaction. The reason is that we selected blastocysts of sufficient quality for biopsy. Wider implications of the findings Using the TLS, we have observed the dynamics of compaction in detail. We found a positive relationship between compaction and blastocyst quality and its association with embryo ploidy. The assessment of compaction is a significant parameter for predicting competent embryos and should be given priority when selecting blastocysts. Trial registration number Not applicable

Premature luteinization is not an uncommon problem in GnRH antagonist cycles. However, the possible effect on IVF outcomes is controversial and it is not unclear when decide the date of administration of human chorionic gonadotropin (hCG) if luteinizing hormone (LH) rises prematurely. Here, we sought to determine whether premature luteinization is associated with adverse IVF outcomes. Retrospective cohort study. A chart review was made of patients who underwent IVF cycle from November 2009 to December 2011. All patients were treated according to the GnRH antagonist protocol, and recombinant FSH administration were only included in this study. Couples with genetic factors and testicular sperm extraction were excluded in this analysis. Subsequent vaginal ultrasound scans and hormonal determinations were performed. Statistical analysis was performed using IBM SPSS 19.0 software. Total 674 cycles were included in this analysis, 231 (35.4%) women were pregnant and premature luteinization occurred in 183 (27.2%) cycles. In normo-responder patients, premature luteinization occurred in 26.7% (129/483), and premature LH surge does not impair IVF outcomes. However, in poor responder patients, the results were different. The incidence of premature luteinization was 27.7% (53/191). Positive hCG rate was significantly lower in the premature luteinization group (18% vs. 2.4%, P=0.01). Only one poor responder got pregnant after early LH rise, but had a spontaneous miscarriage. In addition, the data showed a tendency towards a lower implantation rate for the premature luteinization group, though it was not statistically significant (7.55% vs. 1.25%, P=0.057). Premature luteinization during GnRH antagonist cycles is a frequent event that is associated with adverse IVF outcomes only in the poor responder. Therefore, it needs to be considered earlier administration of hCG in an IVF cycle if LH rises prematurely only in the poor responder.

OBJECTIVE: ICSI was applied and adapted in human ART and became the treatment of choice for infertility factor and repeated IVF failure. Although high successful clinical outcome can be obtained, ICSI might induce a minimal amount of damage of oocytes, and it may cause a slight harmful effect to the microinjected oocytes. Therefore, LA-ICSI, micro-opening of the zona pellucida(ZP) prior to ICSI could be applied as an alternative method to enhance better than clinical outcome. In this study, we have analyzed the efficiency of our modified LA-ICSI, thinning of the ZP prior to ICSI, in patients undergone ICSI. DESIGN: Comparison of clinical outcomes between modified LA-ICSI and conventional-ICSI. MATERIALS AND METHODS: In order to reduce the damage, we have applied approximately 70% of ZP thinning by laser before ICSI and referred to the modified LA-ICSI. From Feb. to Sep., 2009, 880 cycles of ICSI cycles were divided into modified LA-ICSI(106 cycles with 923 MII oocytes) and conventional ICSI(774 cycles with 5156 MII oocytes) groups, and then their clinical outcomes(fertilization, oocytes degeneration, and clinical pregnancy rates and blastocyst development of remaining embryo) were compared. Also, these data were reanalyzed according to patients age group(A: ≤34 yrs, B: ≥35 and ≤38 yrs, C:≥ 39 yrs) and the number of cycles. Statistical significance was analyzed using χ2-test. RESULTS: Fertilization and oocytes degeneration rates of modified LA-ICSI were not different to those of conventional-ICSI(83.3%, 3.1% vs 81.4%, 3.6%, respectively). But, in modified LA-ICSI group, quality of transferred embryos and blastocyst development of remaining embryo were better than those of conventional-ICSI. Also,clinical pregnancy rate of modified LA-ICSI group was significantly higher in all and selected age groups as well as repeated IVF failure group. CONCLUSION: This result suggested that modified LA-ICSI may be applicable to not only old aged and repeated IVF failure groups but also all ICSI patients to improve the chances for pregnancy.

OBJECTIVE: This study was aimed to assess the effect of the endometrial movement on pregnancy achievement in women receiving ovulation induction by clomiphene citrate followed by intrauterine insemination (IUI).DESIGN: Prospective clinical study.MATERIALS AND METHODS: The study population was composed of all infertile couples without any known factor who were to undergo first-time IUI with clomiphene citrate in Fertility Center of CHA University between September 2008 and January 2009. Not only endometrial movements measured using ultrasound (Accuvix, Medison), but also thickness, volume, pattern, and echogenic change of endometrium were analyzed in prediction of pregnancy.RESULTS: The total number of 241 cycles of IUI with 49 intrauterine pregnancies (20.3%) was analyzed, among the total number of 243 cycles of IUI with 51 clinical pregnancies (21.0%) with 2 cycles of tubal pregnancies. Pregnancy was not related to endometrial thickness and endometrial volume, but significantly related to endometrial movement, trilaminar pattern, and hyperechoic change (p<0.05). Endomtrial movements over than 4 times per minute and with weak or moderate intensity were significantly related to pregnancy (p < 0.05). The major direction of endometrial movement was cervicofundal (82.6%), and hence pregnant group showed higher cervicofundal movement rate as well (89.8% vs 75.5%).CONCLUSIONS: Endometrial movement could be a predictor of pregnancy in IUI cycles. Differently from spontaneous cycles, fundocervical endometrial movement which is retrograde for favorable sperm transport can be overcome by IUI procedures. OBJECTIVE: This study was aimed to assess the effect of the endometrial movement on pregnancy achievement in women receiving ovulation induction by clomiphene citrate followed by intrauterine insemination (IUI). DESIGN: Prospective clinical study. MATERIALS AND METHODS: The study population was composed of all infertile couples without any known factor who were to undergo first-time IUI with clomiphene citrate in Fertility Center of CHA University between September 2008 and January 2009. Not only endometrial movements measured using ultrasound (Accuvix, Medison), but also thickness, volume, pattern, and echogenic change of endometrium were analyzed in prediction of pregnancy. RESULTS: The total number of 241 cycles of IUI with 49 intrauterine pregnancies (20.3%) was analyzed, among the total number of 243 cycles of IUI with 51 clinical pregnancies (21.0%) with 2 cycles of tubal pregnancies. Pregnancy was not related to endometrial thickness and endometrial volume, but significantly related to endometrial movement, trilaminar pattern, and hyperechoic change (p<0.05). Endomtrial movements over than 4 times per minute and with weak or moderate intensity were significantly related to pregnancy (p < 0.05). The major direction of endometrial movement was cervicofundal (82.6%), and hence pregnant group showed higher cervicofundal movement rate as well (89.8% vs 75.5%). CONCLUSIONS: Endometrial movement could be a predictor of pregnancy in IUI cycles. Differently from spontaneous cycles, fundocervical endometrial movement which is retrograde for favorable sperm transport can be overcome by IUI procedures.

OBJECTIVE: Robertsonian translocations (RT) are the most common chromosome abnormalities in human with an incidence of 1 in 1000 newborns. A carrier of RT, who is usually phenotypically normal, has a risk of unbalanced gametes resulting in infertility, recurrent abortions, and abnormal child. Then, RT is the most common indication of preimplantation genetic diagnosis (PGD), however, the incidence of abnormal embryos is not determined in general. So, we would like to evaluate the frequencies of abnormal embryos detected by PGD and the good number of embryos for PGD in couples with a RT carrier and to give information for genetic counseling to RT carriers who consider PGD as an option for pregnancy.DESIGN: A retrospective analysis of the results of PGD in couples with a Robertsonian translocation carrier.MATERIALS AND METHODS: 55 cycles of PGD from 32 couples (21 female carriers and 11 male carriers) with a Robertsonian translocation carrier were performed from 2000 to 2007. Single blastomere from each embryo was biopsied on day 3 and fluorescent in situ hybridization was performed with appropriate probe sets.RESULTS: Total 426 blastomeres were tested. One hundred thirty six (31.9%) were normal or balanced, 231 (54.2%) were abnormal and 59 (13.8%) were failure for in situ hybridization. Ninety six embryos out of 136 were transferred in 47 cycles; mean number of transferred embryos is 2.04 per cycle. Total 16 embryos were implanted and 13 babies including two twins were born. Three embryos were aborted spontaneously (2 embryos) or selectively (1 embryos).CONCLUSIONS: In our experience, more than 50% of embryos obtained from Robertsonian translocation carriers showed abnormal chromosome constitution and only about 30% of embryos were normal or balanced chromosome constitution. So, more than 7 embryos per each PGD cycle are needed to obtain two or more normal or balanced embryos. OBJECTIVE: Robertsonian translocations (RT) are the most common chromosome abnormalities in human with an incidence of 1 in 1000 newborns. A carrier of RT, who is usually phenotypically normal, has a risk of unbalanced gametes resulting in infertility, recurrent abortions, and abnormal child. Then, RT is the most common indication of preimplantation genetic diagnosis (PGD), however, the incidence of abnormal embryos is not determined in general. So, we would like to evaluate the frequencies of abnormal embryos detected by PGD and the good number of embryos for PGD in couples with a RT carrier and to give information for genetic counseling to RT carriers who consider PGD as an option for pregnancy. DESIGN: A retrospective analysis of the results of PGD in couples with a Robertsonian translocation carrier. MATERIALS AND METHODS: 55 cycles of PGD from 32 couples (21 female carriers and 11 male carriers) with a Robertsonian translocation carrier were performed from 2000 to 2007. Single blastomere from each embryo was biopsied on day 3 and fluorescent in situ hybridization was performed with appropriate probe sets. RESULTS: Total 426 blastomeres were tested. One hundred thirty six (31.9%) were normal or balanced, 231 (54.2%) were abnormal and 59 (13.8%) were failure for in situ hybridization. Ninety six embryos out of 136 were transferred in 47 cycles; mean number of transferred embryos is 2.04 per cycle. Total 16 embryos were implanted and 13 babies including two twins were born. Three embryos were aborted spontaneously (2 embryos) or selectively (1 embryos). CONCLUSIONS: In our experience, more than 50% of embryos obtained from Robertsonian translocation carriers showed abnormal chromosome constitution and only about 30% of embryos were normal or balanced chromosome constitution. So, more than 7 embryos per each PGD cycle are needed to obtain two or more normal or balanced embryos.

OBJECTIVE: Generally, the creation of small hole in the zona pellucida (ZP) due to intracytoplasmic sperm injection does not influence the cryosurvival of human embryos, because the surface structure of mature oocytes is extremely flexible. However, during assisted hatching (AH) and embryo biopsy for preimplantation genetic diagnosis, the creation of a large hole in the ZP of human embryos does negatively affect cryosurvival. However, our group has suggested that the high cooling rate provided by SN2 may reduce mechanical damage in micromanipulated embryos and results in better embryonic development (Hum Reprod 22;9:2509-14, 2007). So, the aim of this study was to evaluate the effectiveness of different AH methods (partial zona dissection (PZD) and laser pulsed) on the blastocyst cryopreservation.DESIGN: Prospective analysis of different AH methods in ET program using vitrified/warmed blastocysts.MATERIALS AND METHODS: From October 2005 to April 2008, this study was undertaken in patients agreed to participate in the cryopreservation protocol for the extra blastocysts obtained from fresh IVF-ET programs. Patients were divided into 3 groups according to AH methods. Group 1: blastocysts underwent artificial shrinkage (AS) using micro-needle puncture and without AH (n=143 in 56 cycles). Group 2: blastocysts underwent AS and with AH using PZD (n=98 in 40 cycles). Group 3: blastocysts underwent AS and with AH using laser pulse (n=136 in 58 cycles). Dehydration was pre-equilibrated with 1.5M ethylene glycol (EG) for 2.5 min and equilibrated with 5.5M EG and 1.0M sucrose for 20 sec, and then vitrified in slush nitrogen (SN2). The warmed blastocysts in all groups were re-hydrated and cultured in G2 medium with 5% HSA, and then transferred into patients.RESULTS: Survival rates of human blastocysts in the Group 2 and Group 3 after vitrified/warmed were higher than those in the Group 1, respectively (80.6% (79/98), 75.0%(102/136), vs. 51.7% (74/143), p<0.01). Each pregnancy rate of Group 1 was 33.3% (14/42), Group 2 was 58.3% (14/42), and Group3 was 47.2% (24/53). Statistical difference was shown only between Group1 and Group2 (P<0.05).CONCLUSIONS: In vitrification using SN2, the use of AH in blastocysts which underwent AS would be an effective tool for increasing the clinical outcome. OBJECTIVE: Generally, the creation of small hole in the zona pellucida (ZP) due to intracytoplasmic sperm injection does not influence the cryosurvival of human embryos, because the surface structure of mature oocytes is extremely flexible. However, during assisted hatching (AH) and embryo biopsy for preimplantation genetic diagnosis, the creation of a large hole in the ZP of human embryos does negatively affect cryosurvival. However, our group has suggested that the high cooling rate provided by SN2 may reduce mechanical damage in micromanipulated embryos and results in better embryonic development (Hum Reprod 22;9:2509-14, 2007). So, the aim of this study was to evaluate the effectiveness of different AH methods (partial zona dissection (PZD) and laser pulsed) on the blastocyst cryopreservation. DESIGN: Prospective analysis of different AH methods in ET program using vitrified/warmed blastocysts. MATERIALS AND METHODS: From October 2005 to April 2008, this study was undertaken in patients agreed to participate in the cryopreservation protocol for the extra blastocysts obtained from fresh IVF-ET programs. Patients were divided into 3 groups according to AH methods. Group 1: blastocysts underwent artificial shrinkage (AS) using micro-needle puncture and without AH (n=143 in 56 cycles). Group 2: blastocysts underwent AS and with AH using PZD (n=98 in 40 cycles). Group 3: blastocysts underwent AS and with AH using laser pulse (n=136 in 58 cycles). Dehydration was pre-equilibrated with 1.5M ethylene glycol (EG) for 2.5 min and equilibrated with 5.5M EG and 1.0M sucrose for 20 sec, and then vitrified in slush nitrogen (SN2). The warmed blastocysts in all groups were re-hydrated and cultured in G2 medium with 5% HSA, and then transferred into patients. RESULTS: Survival rates of human blastocysts in the Group 2 and Group 3 after vitrified/warmed were higher than those in the Group 1, respectively (80.6% (79/98), 75.0%(102/136), vs. 51.7% (74/143), p<0.01). Each pregnancy rate of Group 1 was 33.3% (14/42), Group 2 was 58.3% (14/42), and Group3 was 47.2% (24/53). Statistical difference was shown only between Group1 and Group2 (P<0.05). CONCLUSIONS: In vitrification using SN2, the use of AH in blastocysts which underwent AS would be an effective tool for increasing the clinical outcome.

Pre-eclampsia is a disease in pregnancy which contributes significantly to maternal and fetal mortality and morbidity. Moreover, ART-conceived twin pregnancies (Daniel, 2000) and pregnancies after oocyte donation (Yang, 2001) are at higher risk for pre-eclampsia. The etiologic factors of pre-eclampsia have not been completely clear, although evidence supports involvement of genetic, immune, angiogenic, and other mechanisms. To clarify the possible relation between gene expression and the placental dysfunction in pre-eclampsia, we examined the expression of regulated genes in placenta tissues. Retrospective study. Patients This study was performed using frozen placenta tissues obtained by spontaneous vaginal deliveries of the patient and control woman. First-strand cDNA Synthesis and ACP (Annealing Control Primer)-based GeneFishing™ PCR Extracted total RNAs were used for the synthesis of first-strand cDNAs by RT. Differentially expressed genes were screened by ACP-based PCR method (Kim et al., 2004) using the GeneFishing™ DEG (Differentially Expressed Gene) kits (Seegene, South Korea). After second-strand DNA synthesis was completed, the second-stage PCR amplification protocol was performed. Cloning and Sequencing The differentially expressed bands were extracted from the gel by using the GENCLEAN®II Kit (Q-BIO gene, USA), and directly cloned into a TOPO TA cloning vector (Invitrogen, USA). The cloned plasmids were sequenced with ABI PRISM®3100 Genetic Analyzer (Applied Biosystems, USA). Seventy-two DEG were found by the GeneFishing™ DEG full-screening. It means 72 genes were expressed differently at pre-eclampsia and normal placenta. After cloning and sequencing of these genes, we isolated 8 genes. Three genes - nectin-3, chorionic somatomammotropin (CSH), 12qBAC RP11-49G17 - were high regulated in normal placenta. Five genes - interleukin I receptor, interferon induced transmembrane protein, eosinophil granule major basic protein, myofibrillogenesis regulator 1, and zinc finger calcium binding protein - were high expressed in pre-eclampsia placenta. In this study, reduced CSH1 and Nectin-3 expression was detected in placenta of the patient. These two genes might be closely related this disease. Nectins are Ig superfamily adhesion molecules, nectin-3 gets trans-heterophilic interactions either nectin-1 or nectin-2, and the complex may play a role in cell to cell adhesion, regulates the physiology of epithelial or endothelial cells in normal placenta. The protein of CSH1 gene, called placental lactogen, is a member of the somatotropin/prolactin family of hormones and plays an important role in growth control. CSH1 gene’s mutations result in placental lactogen deficiency and Silver-Russell syndrome. Pre-eclampsia decreases utero-placental perfusion, which has been placed the fetus at high risk for problems like preterm birth. Pre-eclampsia has been believed to be related to one or more genes and the search for genetic factors predisposing to pre-eclampsia has been intense. We found several genes may be related placental dysfunction in pre-eclampsia. The functional study of these genes should be followed.

GDM (gestational diabetes mellitus) is a one of complications after ART pregnancies. Women with GDM are at risk of subsequent development of overt diabetes, mainly non-insulin-dependent mellitus, later in life. This is a real public health problem that deserves investigation of phenotypic and genotypic predisposing factors. Although susceptibility to GDM has been suggested to have genetic determinant, the precise genetic basis for GDM is yet unclear. The purpose of this experiment was to investigate areas of mtDNA assumed to be of special interest with regard to GDM patients and to study the relationship between SNP (Single Nucleotide Polymorphism) in HV1/ HV2. Therefore, development of efficient SNP assay protocols for HV1 and HV2 would find susceptibility to GDM as well as to pathogenesis of GDM. Prospective observational study with retrospective analysis Peripheral blood samples were collected from patients with GDM (mean age, 31.7±4.3) who were under the inclusion criteria for GDM and from 80 controls (29.4±4.8). Genomic DNA used to be amplified, sequenced and compared with those recorded in the mitochondrial databank (http: //www.mitomap.org). Statistical analysis was assessed by Chi-square and Fisher’s exact (p < 0.05). The result of this analysis is shown in table. Most of the mutations in both GDM and control sequences were single base substitutions and most of these were transitions. In both regions, transitions were 92.1%, transversions 2.8%, insertions 0.4%, and deletions 4.7%. Tabled 1 In this study, we analyzed the SNP in HV1 and HV2, that showed higher mutation rate in GDM patients. It has been demonstrated that some mtDNA mutations are involved in the pathogenesis of GDM. An A3243G point mutation (tRNA-Leu (UUR)) and many other mutations in the HV regions have been described in association with GDM. Likewise, significantly lower mtDNA copy number in GDM patients than in control group has been reported (2004 ASRM Kim et al.). We have found a novel SNP in HV2 of noncoding regions in GDM patient. The transition, A290G, in the mTF1 binding site has not been described previously. In terms of mTF1 binding site, mitochondrial transcription factor is an important regulator of both transcription and replication of mtDNA. Moreover, mitochondrial transcription factor regulates mtDNA copy number. In noncoding areas, 92.13 % of the GDM patients and 84.56 % of the group had above transition. The statistically significant SNPs detected in this study are known from previous studies: A235G in the Conserved sequence block1 and T152C in the H-strand origin. We have also been given by its high mutation rate (22.86% in A235G and 34.28% in T152C). It may serve as sensitive diagnostic markers with its mutation level being indicative of GDM. Furthermore, polymorphisms of mtDNA may play a role in patients with GDM.

To improve the viability and quality of oocytes after vitrification, we have introduced several changes, new type of vitrification vehicle and mediator, gold grid and slush-liquid nitrogen (S-LN2), applied in order to elevate cooling speed for reducing ice crystal formation. Gold grid can provide extremely high heat conductivity. And by applying negative pressure with a vacuum, LN2 will freeze and convert into a slush state. Slush-LN2 has a lower internal temperature of 201°C without vaporization. Since it may offer high-speed cooling rates, it may be possible to increase the survival rate as well as other characteristics. In fact, a higher cooling rate resulting from the use of gold grid and slush-LN2 is beneficial for the survival and fertilization of human mature oocytes after vitrification. From these results, our modified vitrification system would be a promising technique to effectively reserve human oocytes.

Oocytes meiotic cell cycle progression is thought to be orchestrated by protein phosphorylation-dephosphorylation cascades. MPF (maturation- or metaphase-promoting factor) and MAP (microtubule-associated or mitogen-activated protein) kinase are two key molecules involved in this process. MPF plays a pivotal role in driving the oocyte meiotic cell cycle progression and MAP kinase plays an important role in controlling chromatin and microtubule behavior. However, the role of MAP kinase phosphorylation during maturation, fertilization, and early embryonic development has not been studied in humans. We investigated change of MAP kinase phosphorylation pattern of oocytes during in vitro maturation and early embryonic development in humans. Experimental study utilizing human oocytes and embryos. Human oocytes and embryos were collected from consented stimulated IVF patients and divided into 4 groups. Group 1 : immature oocytes (GV stage oocytes), Group 2 : unfertilized mature oocyte (MII stage oocytes), Group 3 : fertilized embryos (tripronuclei embryos, 3PN), Group 4 : cleavaged embryos (2-cell and 8-cell stage embryos). Immature oocytes were retrieved 34–36 hours after hCG administration and collected for analysis. Twenty hours after insemination, the oocytes were examined for the presence of pronuclei and then collected the unfertilized oocytes. The tripronucleated embryos were obtained 18–24 hours after retrieval. Two-cell and 8-cell embryos were cultured in P-1 medium and collected after 2 and 3 days. Four groups used for detection of MAP kinase phosphorylation assay. MAP kinase phosphorylation was revealed by western immunoblotting. Proteins extracted from oocytes and embryos, electrophoresised, transferred to the nitrocellulose membrane, the membrane was incubated with primary anti-phosphorylated MAP kinase antibody followed by biotynylated second antibody. After incubation for 1 min in the ECL detection solution, we exposed the membrane to autoradiography film for detection. This experiment was repeated three times. MAP kinase was not phosphorylated at the GV stage oocytes but mature oocytes contained two forms of MAP kinase, ERK 1, ERK 2 that become phosphorylated. After insemination and culture, MAP kinase phosphorylation of 3PN embryos, 2-cell embryos was still present in a phosphorylated form. However, the MAP kinase was dephosphorylated at the 8-cell stage embryos. This study suggests that phosphorylation/dephosphorylation event of MAP kinase occur stage-specific during oocyte maturation and early embryonic development in humans. Further studies of the mechanism of MAP kinase role are necessary for ART program.

Objective: The sperm mitochondria and the majority of cytoplasmic mitochondria cannot be detected in advanced embryonic stages except in unusual situations such as inter-specific hybrids and artificial cytoplasmic constructs. The exact mechanism of elimination of sperm mitochondria is unknown, but now it is can be mediated by ubiqutin. The procedure of intracytoplasmic sperm injection (ICSI) results in an acrosome which is present in the cytoplasm rather than being lost. Abnormal spermatozoa, which harbor mutation in mitochondrial DNA (mtDNA) and especially common deletion, is a safe issue that should be considered when performing ICSI. The purpose of this study was to compare the ratio of the amount of deletion taken from ICSI induced pregnancy amniocytes to the amounts determined in normal pregnancy amniocytes. PCR assays were accomplished on each study group to formulate the ratio.Design: Using a serial dilution PCR approach, prospective cultures of amniocytes from ICSI and normal pregnancy were studied to measure the amount of deletion.Materials and Methods: The subjects consisted of 106 fetuses of ICSI pregnancy and 117 fetuses of non-ICSI pregnancy that were confirmed with normal karyotypes. Genomic DNA was isolated from amniotic fluid after 15–23 week of gestation in fetuses. Before quantification, common deletion was examined by nested PCR with the primer (F1: nt8225–8247, R1: nt13574–13511, R2: nt13729–13706) to compare the frequency of common deletion in ICSI and non-ICSI samples. A competitive PCR assay was performed using serial dilutions of the competitor construct in conjunction with a constant amount 100ng of DNA (F2: nt8285–8308, R3: nt13506–13483). Standard curves for quantifying the results were drawn and statistical significance of the differences was evaluated by Fisher’s exact test and the Chi-square test.Results: The mean maternal age of ICSI and non ICSI amniocytes was 33.94 and 32.49 years old respectively. After nested PCR for detection of deletion, there was a statistically significant difference in the frequency of common deletion (Fisher’s exact test: P = 0.0001) in ICSI and non ICSI pregnancy (81.1%:33.3%). A competitive PCR resulted as 0.047779 vs. 0.00235 in ICSI and non ICSI amniocytes. A statistically significant difference (Chi-square test: p < 0.0001) resulted by doing these test.Conclusion: Inheritance of the mitochondria is still under investigation. However, it is believed that in mammals the mitochondria are inherited from the oocyte during fertilization. In further work, the zygote cytoplasm has a species-specific mechanism that recognizes and eliminates sperm mitochondria, on the basis of nuclear DNA encoded proteins in the sperm midpiece, not on the mtDNA itself, nor on the proteins it encodes. The procedures of ICSI bypassing the induction of capacitation and acrosome reaction by a motile spermatozoon for oocyte penetration, the possibility exists that ICSI may transmit genetic abnormalities. In this regard, the ICSI procedure might result in iatrogenic induction of mitochondrial mutation that might evade destruction. According to our result, significantly higher amounts of common deletion in ICSI pregnancy than existed in non ICSI pregnancy, which means ICSI differs from natural sterilization in terms of bypassing some natural process, including natural selection of mtDNA deletion. However, clinical disease in humans is generally not observed until the mitochondrial function is reduced by more than 80%. Objective: The sperm mitochondria and the majority of cytoplasmic mitochondria cannot be detected in advanced embryonic stages except in unusual situations such as inter-specific hybrids and artificial cytoplasmic constructs. The exact mechanism of elimination of sperm mitochondria is unknown, but now it is can be mediated by ubiqutin. The procedure of intracytoplasmic sperm injection (ICSI) results in an acrosome which is present in the cytoplasm rather than being lost. Abnormal spermatozoa, which harbor mutation in mitochondrial DNA (mtDNA) and especially common deletion, is a safe issue that should be considered when performing ICSI. The purpose of this study was to compare the ratio of the amount of deletion taken from ICSI induced pregnancy amniocytes to the amounts determined in normal pregnancy amniocytes. PCR assays were accomplished on each study group to formulate the ratio. Design: Using a serial dilution PCR approach, prospective cultures of amniocytes from ICSI and normal pregnancy were studied to measure the amount of deletion. Materials and Methods: The subjects consisted of 106 fetuses of ICSI pregnancy and 117 fetuses of non-ICSI pregnancy that were confirmed with normal karyotypes. Genomic DNA was isolated from amniotic fluid after 15–23 week of gestation in fetuses. Before quantification, common deletion was examined by nested PCR with the primer (F1: nt8225–8247, R1: nt13574–13511, R2: nt13729–13706) to compare the frequency of common deletion in ICSI and non-ICSI samples. A competitive PCR assay was performed using serial dilutions of the competitor construct in conjunction with a constant amount 100ng of DNA (F2: nt8285–8308, R3: nt13506–13483). Standard curves for quantifying the results were drawn and statistical significance of the differences was evaluated by Fisher’s exact test and the Chi-square test. Results: The mean maternal age of ICSI and non ICSI amniocytes was 33.94 and 32.49 years old respectively. After nested PCR for detection of deletion, there was a statistically significant difference in the frequency of common deletion (Fisher’s exact test: P = 0.0001) in ICSI and non ICSI pregnancy (81.1%:33.3%). A competitive PCR resulted as 0.047779 vs. 0.00235 in ICSI and non ICSI amniocytes. A statistically significant difference (Chi-square test: p < 0.0001) resulted by doing these test. Conclusion: Inheritance of the mitochondria is still under investigation. However, it is believed that in mammals the mitochondria are inherited from the oocyte during fertilization. In further work, the zygote cytoplasm has a species-specific mechanism that recognizes and eliminates sperm mitochondria, on the basis of nuclear DNA encoded proteins in the sperm midpiece, not on the mtDNA itself, nor on the proteins it encodes. The procedures of ICSI bypassing the induction of capacitation and acrosome reaction by a motile spermatozoon for oocyte penetration, the possibility exists that ICSI may transmit genetic abnormalities. In this regard, the ICSI procedure might result in iatrogenic induction of mitochondrial mutation that might evade destruction. According to our result, significantly higher amounts of common deletion in ICSI pregnancy than existed in non ICSI pregnancy, which means ICSI differs from natural sterilization in terms of bypassing some natural process, including natural selection of mtDNA deletion. However, clinical disease in humans is generally not observed until the mitochondrial function is reduced by more than 80%.