Lifestyle and fertility-specific quality of life affect reproductive outcomes in couples undergoing in vitro fertilization.

Objective: A Mediterranean dietary pattern, sleeping habits, physical activity, and lifestyle appear to affect reproductive health. There are few reports about whether fertility-specific quality of life (QOL) is linked to infertility treatment outcomes. The aim of this study is to investigate when lifestyle factors and fertility-specific QOL are comprehensively considered, which factors influence assisted reproductive technology (ART) outcomes. Methods: This prospective cohort includes 291 women undergoing a first ART treatment at multiple centers in Japan and was designed to evaluate the influence of diet, physical activity, sleeping pattern, computer use duration, and fertility-specific quality of life tool (FertiQoL) score on ART treatment outcomes using a questionnaire. The primary endpoint was the good-quality blastocyst rate per oocyte retrieval and the secondary endpoints were a positive pregnancy test and gestational sac (GS) detection. Results: The good-quality blastocyst rate per oocyte retrieval tended to be negatively associated with frequent fish consumption. After all embryo transfer (ET) cycles, a positive pregnancy test tended to be positively associated with longer sleep and longer computer use (OR = 1.6, 95% CI = 0.9-2.7 and OR = 1.7, CI = 1.0-2.8, respectively) and negatively associated with a smoking partner (OR = 0.6, CI = 0.3-1.0). GS detection was positively and significantly associated with frequent olive oil intake and longer computer use (OR = 1.7, CI = 1.0-3.0 and OR = 1.7, CI = 1.0-3.0, respectively). After ET cycles with a single blastocyst, a positive pregnancy test was positively and significantly associated with longer computer use (OR = 2.0, CI = 1.1-3.7), while GS detection was significantly more likely in women with longer computer use (OR = 2.1, CI = 1.1-3.8) and tended to be more likely in women with a higher FertiQoL Total scaled treatment score (OR = 1.8, CI = 1.0-3.3). p < 0.05 was considered statistically significant and 0.05 ≤ p <0.01 as tendency. Conclusions: Olive oil may be an important factor in dietary habits. Fertility-specific QOL and smoking cessation guidance for partners are important for infertile couples.

Impact of Lactobacillus in the uterine microbiota on in vitro fertilization outcomes.

Abundant intrauterine Lactobacillus is associated with good in vitro fertilization (IVF) outcomes; however, whether specific species of Lactobacillus have any benefit remains unclear. So we examine the effect of Lactobacillus on the clinical outcomes of IVF at the species level. Uterine microbiota were classified as either Lactobacillus-dominant (LD) or non-Lactobacillus-dominant. In the LD group, we further investigated the clinical results for each Lactobacillus species and evaluated them in relation to IVF outcomes. In Uterine microbiome analysis, Lactobacillus was the most abundant, with the four species of L. crispatus, L. iners, L. gasseri, and L. jensenii accounting for the great majority. We compared the clinical outcomes of single frozen-thawed embryo transfer conducted by Lactobacillus species and found that the implantation rate was lowest in those in whom L. iners was dominant. This study is the first to conduct a species-level analysis of the uterine microbiota and report on a detailed investigation of Lactobacillus, which was believed to be particularly helpful for pregnancy.

Sperm and Oocyte Chromosomal Abnormalities.

Gametogenesis, the process of producing gametes, differs significantly between oocytes and sperm. Most oocytes have chromosomal aneuploidies, indicating that chromosomal aberrations in miscarried and newborn infants are of oocyte origin. Conversely, most structural anomalies are of sperm origin. A prolonged meiotic period caused by increasing female age is responsible for an increased number of chromosomal aberrations. Sperm chromosomes are difficult to analyze because they cannot be evaluated using somatic cell chromosome analysis methods. Nevertheless, researchers have developed methods for chromosome analysis of sperm using the fluorescence in situ hybridization method, hamster eggs, and mouse eggs, allowing for the cytogenetic evaluation of individual sperm. Reproductive medicine has allowed men with severe spermatogenic defects or chromosomal abnormalities to have children. However, using these techniques to achieve successful pregnancies results in higher rates of miscarriages and embryos with chromosomal abnormalities. This raises questions regarding which cases should undergo sperm chromosome analysis and how the results should be interpreted. Here, we reviewed clinical trials that have been reported on oocyte and sperm chromosome analyses. Examination of chromosomal abnormalities in gametes is critical in assisted reproductive technology. Therefore, it is necessary to continue to study the mechanism underlying gametic chromosomal abnormalities.

Clinical application of long-read nanopore sequencing in a preimplantation genetic testing pre-clinical workup to identify the junction for complex Xq chromosome rearrangement-related disease.

OBJECTIVE: Xq chromosome duplication with complex rearrangements is generally acknowledged to be associated with neurodevelopmental disorders, such as Pelizaeus-Merzbacher disease (PMD) and MECP2 duplication syndrome. For couples who required a PGT-M (pre-implantation genetic testing for monogenic disease) for these disorders, junction-specific PCR is useful to directly detect pathogenic variants. Therefore, pre-clinical workup for PGT-M requires the identification of the junction of duplicated segments in PMD and MECP2 duplication syndrome, which is generally difficult. METHODS: In this report, we used nanopore long-read sequencing targeting the X chromosome using an adaptive sampling method to identify breakpoint junctions in disease-causing triplications. RESULTS: By long-read sequencing, we successfully identified breakpoint junctions in one PMD case with PLP1 triplication and in another MECP2 triplication case in a single sequencing run. Surprisingly, the duplicated region involving MECP2 was inserted 45 Mb proximal to the original position. This inserted region was confirmed by FISH analysis. With the help of precise mapping of the pathogenic variant, we successfully re-established STR haplotyping for PGT-M and avoided any potential misinterpretation of the pathogenic allele due to recombination. CONCLUSION: Long-read sequencing with adaptive sampling in a PGT-M pre-clinical workup is a beneficial method for identifying junctions of chromosomal complex structural rearrangements.

Analysis of clinical outcomes and meiotic segregation modes following preimplantation genetic testing for structural rearrangements using aCGH/NGS in couples with balanced chromosome rearrangement.

Purpose: To retrospectively evaluate the effectiveness of PGT-SR by array comparative genomic hybridization (aCGH) or next-generation sequencing (NGS) in preventing recurrent miscarriages. Methods: Thirty one couples with balanced translocation who underwent 68 PGT-SR cycles between 2012 and 2020 were evaluated. A total of 242 blastocysts were biopsied for aCGH or NGS. The genetically transferable blastocysts were transferred in the subsequent frozen-thawed single embryo transfer cycle. Results: The genetically transferable blastocyst rate was 21.2% (51/241). Thirty five genetically transferable blastocysts were transferred into the uterine cavity. The clinical pregnancy rate was 57.1% (20/35), and the ongoing pregnancy rate was 100.0% (20/20). The incidence of interchromosomal effect (ICE) was influenced by ovarian stimulation protocol, female age, and carrier's gender, but dependent on the types of balanced translocation carriers. Furthermore, there was no significant difference in meiotic segregation modes in ovarian stimulation protocols and carrier's gender. Interestingly, the incidence of adjacent-1 segregation in ≧40 years group increased significantly compared with <35 years group. Conclusions: For the first time in Japan, we show the effectiveness of PGT-SR using aCGH or NGS, which enables comprehensive analysis of chromosomes, in the prevention of recurrent miscarriages. Furthermore, our results may support better genetic counseling of balanced translocation carriers for PGT-SR cycles.

Usefulness of combined NGS and QF-PCR analysis for product of conception karyotyping.

Purpose: Since chromosomal abnormalities can be detected in more than half of miscarriages, cytogenetic testing of the product of conception (POC) can provide important information when preparing for a subsequent pregnancy. Conventional karyotyping is the common diagnostic method for a POC but can be problematic due to the need for cell culture. Methods: We here conducted shallow whole-genome sequencing (sWGS) using next-generation sequencing (NGS) for alternative POC cytogenomic analysis. Since female euploidy samples can include 69,XXX triploidy, additional QF-PCR was performed in these cases. Results: We here analyzed POC samples from miscarriages in 300 assisted reproductive technology (ART) pregnancies and detected chromosomal abnormalities in 201 instances (67.0%). Autosomal aneuploidy (151 cases, 50.3%) was the most frequent abnormality, consistent with prior conventional karyotyping data. Mosaic aneuploidy was detected in seven cases (2.0%). Notably, the frequency of triploidy was 2.3%, 10-fold lower than the reported frequency in non-ART pregnancies. Structural rearrangements were identified in nine samples (3%), but there was no case of segmental mosaicism. Conclusions: These data suggest that NGS-based sWGS, with the aid of QF-PCR, is a viable alternative karyotyping procedure that does not require cell culture. This method could also assist with genetic counseling for couples who undergoes embryo selection based on PGT-A data.

Effects of exposure to methylglyoxal on sperm motility and embryonic development after fertilization in mice.

Methylglyoxal (MG) is a precursor for the generation of endogenous advanced glycation end-products involved in various diseases, including infertility. The present study evaluated the motility and developmental competence after in vitro fertilization of mouse sperm which were exposed to MG in the capacitation medium for 1.5 h. Sperm motility was analyzed using an SQA-V automated sperm quality analyzer. Intracellular reactive oxygen species (ROS), membrane integrity, mitochondrial membrane potential, and DNA damage were assessed using flow cytometry. The matured oocytes were inseminated with MG-exposed sperm, and subsequently, the fertilization and embryonic development in vitro were evaluated in vitro. The exposure of sperm to MG did not considerably affect the swim-up of sperm but resulted in a deteriorated sperm motility in a concentration-dependent manner, which was associated with a decreased mitochondrial activity. However, these effects was not accompanied by obvious ROS accumulation or DNA damage. Furthermore, MG diminished the fertilization rate and developmental competence, even after normal fertilization. Collectively, a short-term exposure to MG during sperm capacitation had a critical impact on sperm motility and subsequent embryonic development after fertilization. Considering that sperm would remain in vivo for up to 3 days until fertilization, our findings suggest that sperm can be affected by MG in the female reproductive organs, which may be associated with infertility.

Treatment with Laevo (L)-carnitine reverses the mitochondrial function of human embryos.

PURPOSE: Laevo (l)-carnitine plays important roles in reducing the cytotoxic effects of free fatty acids by forming acyl-carnitine and promoting beta-oxidation, leading to alleviation of cell damage. Recently, the mitochondrial functions in morula has been shown to decrease with the maternal age. Here, we assessed the effect of l-carnitine on mitochondrial function in human embryos and embryo development. METHODS: To examine the effect of L-carnitine on mitochondrial function in morulae, 38 vitrified-thawed embryos at the 6-11-cell stage on day 3 after ICSI were donated from 19 couples. Each couple donated two embryos. Two siblings from each couple were divided randomly into two groups and were cultured in medium with or without 1 mM L-carnitine. The oxygen consumption rates (OCRs) were measured at morula stage. The development of 1029 zygotes cultured in medium with or without L-carnitine was prospectively analyzed. RESULTS: Addition of L-carnitine to the culture medium significantly increased the OCRs of morulae and improved the morphologically-good blastocyst formation rate per zygote compared with sibling embryos. Twenty healthy babies were born from embryos cultured in L-carnitine-supplemented medium after single embryo transfers. CONCLUSION(S): L-carnitine is a promising culture medium supplement that might be able to counteract the decreased mitochondrial function in human morula stage embryos.

Mitochondrial oxygen consumption rate of human embryos declines with maternal age.

PURPOSE: The fertility of women decreases with age because of factors such as an increased incidence of aneuploidies and-possibly-decreased mitochondrial activity in oocytes. However, the relationship between maternal aging and mitochondrial function of their embryos remains unknown. Here, we assessed the relationship between maternal age and mitochondrial functions in their oocytes and embryos METHODS: The relationships between maternal age and oxygen consumption rates (OCRs), mitochondrial DNA (mtDNA) copy numbers, or blastocyst development was investigated using 81 embryos donated from 63 infertility couples. The developmental rates from morulae to blastocysts were retrospectively analyzed using data of 105 patients. RESULTS: The OCRs of morulae decreased with maternal age (r2 = 0.48, P < 0.05) although there were no relationships between maternal age and mtDNA copy number in any stages. The more oxygen consumed at the morula stage, the shorter time was required for embryo development to the mid-stage blastocyst (r2 = 0.236, P < 0.05). According to the clinical data analysis, the developmental rate from morulae to blastocysts decreased with maternal age (P < 0.05, < 37 years, 81.1%, vs. ≥ 37 years, 64.1%). CONCLUSIONS: The data of the present study revealed that mitochondrial function at the morula stage of human embryos decreased with their maternal age and a decrease of mitochondrial function led to slow-paced development and impaired developmental rate from morulae to blastocysts.

Use of a vaginal probiotic suppository and antibiotics to influence the composition of the endometrial microbiota.

The aim of the present study was to determine the status of the endometrial microbiota in patients with repeated implantation failure (RIF) and to assess treatment strategies for nondominant Lactobacillus (NLD) cases. A total of 392 patients with RIF were enrolled in this prospective cohort study (UMIN-CTR 000038582) and underwent endometrial microbiota analysis. Patients diagnosed with NLD were treated with a combination of oral and vaginal probiotics or oral prebiotics and antibiotics. The outcome was evaluated through re-analysis of the endometrial microbiota following treatment, and the results are presented as cure rates. NLD represented 44.9 % of the total endometrial microbiota in patients with RIF. The most commonly detected bacterium was Gardnerella vaginalis. The cure rates in the oral probiotics + oral prebiotics, antibiotics, oral probiotics + oral prebiotics + antibiotics, vaginal probiotic suppository, and vaginal probiotic suppository + antibiotics groups were 29.5, 33.33, 33.33, 43.6, and 78.6 %, respectively. Significant improvements were noted in the vaginal probiotic suppository + antibiotics group. Moreover, we revealed that approximately half of patients with RIF had NLD. Thus, the combination of a vaginal probiotic suppository and antibiotics may represent an effective treatment for NLD cases.

Prc1-rich kinetochores are required for error-free acentrosomal spindle bipolarization during meiosis I in mouse oocytes.

Acentrosomal meiosis in oocytes represents a gametogenic challenge, requiring spindle bipolarization without predefined bipolar cues. While much is known about the structures that promote acentrosomal microtubule nucleation, less is known about the structures that mediate spindle bipolarization in mammalian oocytes. Here, we show that in mouse oocytes, kinetochores are required for spindle bipolarization in meiosis I. This process is promoted by oocyte-specific, microtubule-independent enrichment of the antiparallel microtubule crosslinker Prc1 at kinetochores via the Ndc80 complex. In contrast, in meiosis II, cytoplasm that contains upregulated factors including Prc1 supports kinetochore-independent pathways for spindle bipolarization. The kinetochore-dependent mode of spindle bipolarization is required for meiosis I to prevent chromosome segregation errors. Human oocytes, where spindle bipolarization is reportedly error prone, exhibit no detectable kinetochore enrichment of Prc1. This study reveals an oocyte-specific function of kinetochores in acentrosomal spindle bipolarization in mice, and provides insights into the error-prone nature of human oocytes.

Analysis of 122 triplet and one quadruplet pregnancies after single embryo transfer in Japan.

RESEARCH QUESTION: What is the prevalence of triplet and quadruplet pregnancies after single embryo transfer (SET) in Japan. DESIGN: A retrospective observational study was conducted on 274,605 pregnancies after 937,848 SET cycles in registered assisted reproductive technology (ART) data from the Japanese ART national registry database between 2007 and 2014. A questionnaire survey of ART centres was also conducted. Data on pregnancies with embryo division into three or more after SET were analysed. RESULTS: According to the Japanese ART national registry database, SET resulted in 109 triplet pregnancies (0.04% of pregnancies), and the questionnaire reports from 31 centres revealed 33 triplet and one quadruplet pregnancies. After exclusion of 20 duplicated cases, 122 triplet and one quadruplet pregnancies included 46 monochorionic (one gestational sac [37.4%]), 18 dichorionic (two gestational sacs [14.6%]) and 59 trichorionic pregnancies (three gestational sacs [48.0%]). Compared with singleton pregnancies, patients with monozygotic triplet or quadruplet pregnancies were less frequently diagnosed with unexplained infertility (P = 0.004), more often received gonadotrophin injections for ovarian stimulation in 39 cases with information available (P = 0.021) and underwent more blastocyst transfers and assisted hatching (P = 0.002 and P < 0.001, respectively). The proportion of live birth, defined as at least one baby born, excluding induced abortion, was 64.6% (73/116 pregnancies) of monozygotic triplet or quadruplet pregnancies. CONCLUSIONS: Combined Japanese ART national registry and survey data revealed 122 triplet and one quadruplet pregnancies, the majority after cryopreserved embryo transfer. Most were conceived after blastocyst transfer and often after assisted hatching, which are potential risk factors for zygotic splitting.

Sexual Activity of Patients Undergoing Testicular Sperm Extraction.

INTRODUCTION: For couples who wish for a baby, sexual activity (through intercourse) is the only way to conceive naturally. However, the sexual activity of men undergoing testicular sperm extraction (TESE) and factors that influence it among affected couples are not clearly understood. AIM: To examine sexual activity among infertile men undergoing TESE and to identify factors that influenced it. METHODS: Married Japanese male patients undergoing TESE were included. Sexual activity during the previous month was surveyed using a questionnaire, and potential predictive factors influencing sexual activity were examined. MAIN OUTCOME MEASURES: Two hundred twenty-six married men who answered a questionnaire regarding sexual frequency were reviewed retrospectively. RESULTS: The patient mean age was 35.8 ± 7.5 years (range 23-67) and the mean age of their partners was 32.9 ± 4.5 years (range 23-44). Most couples (n = 193) were in their first marriage, and 33 were remarried. The mean marriage duration was 52.6 ± 42.2 months (range 1-192). Microdissection TESE and conventional TESE were performed for 152 and 74 patients, respectively. Overall, the mean sexual activity during the last month was 3.6 ± 2.6 times (range 0-15). Marriage duration was negatively correlated with the frequency of sexual activity (r = -0.23; P < .01). There were no correlations between sexual activity and patient age, partner age, marriage type (first marriage or remarried), testicular volume, or serum total testosterone concentration. Regression analysis showed that marriage duration (odds ratio = 1.01; 95% CI, 1.003-1.019; P = .009) and testicular volume (odds ratio = 0.94; 95% CI, 0.88-0.99; P = .033) were significant predictors of low sexual activity (0-1 times/month). CONCLUSION: The sexual activity of patients undergoing TESE was almost the same as reported previously. Patient age, partner age, testicular volume, and serum total testosterone concentration had no correlation with the reported frequency of sexual activity. Taniguchi H, Matsuda T, Nakaoka Y, et al. Sexual Activity of Patients Undergoing Testicular Sperm Extraction. Sex Med 2019;8:30-35.

Oral administration of l-carnitine improves the clinical outcome of fertility in patients with IVF treatment.

Age-dependent decline of mitochondrial function has been proposed to be a main cause of decline of embryo quality. Then, l-carnitine plays important roles in reducing the membranous toxicity of free-fatty acids by forming acyl-carnitine and promoting ß-oxidation, preventing cell damage. Recent research revealed that l-carnitine played important roles in vitro in oocyte growth, oocyte maturation and embryo development. However, such beneficial effects of l-carnitine in vivo have yet to be verified. The effect of oral l-carnitine supplementation on embryo quality and implantation potential was examined. A total of 214 patients were included in this study. They all previously received in vitro fertilization-embryo transfer (IVF-ET) and failed to conceive. Then they were administered l-carnitine for 82 days on average and underwent IVF-ET again. There were no significant differences in the total number of retrieved oocytes, and their maturation and fertilization rates between before and after l-carnitine administration. The quality of embryos on Days 3 and 5 after insemination was improved following l-carnitine administration (p < .05) in cycles after l-carnitine administration compared with previous cycles. Healthy neonates were born after IVF-ET following l-carnitine administration. Our data suggested that oral administration of l-carnitine to fertility patients improved the developmental competence of their oocytes after insemination.

Age-specific serum anti-Müllerian hormone concentration in Japanese women and its usefulness as a predictor of the ovarian response.

Purpose: To compare the ovarian response predictive ability of anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), and estradiol (E2) and to determine the age-specific distribution of serum AMH concentrations of Japanese women. Methods: This was a multicenter (four-site), observational, analytic, cross-sectional Japanese study consisting of two parts: Study 1 (the prediction of the ovarian response of 236 participants who were undergoing controlled ovarian stimulation [COS]) and Study 2 (the distribution of the AMH concentration with an assay of 417 healthy women who were aged 20-49 years and who had normal menstrual cycles). Results: The AMH had a significantly higher predictive value for the normal and high responders than FSH and E2 as a stronger correlation between the ovarian response and AMH was observed than for FSH and E2. The serum AMH concentration decreased proportionally with age. Conclusion: The AMH concentration correlated well with the oocyte count in the patients who were undergoing COS for in vitro fertilization and was shown to predict the risk of ovarian hyperstimulation syndrome among these patients.

Optimum culture duration for growing oocytes to attain meiotic and fertilization competence.

To determine the optimum culture duration for porcine growing oocytes (GOs) to attain maturation competence, we examined the meiotic competence, chromatin configuration, and fertilization ability of porcine oocytes obtained from early antral follicles and cultured for 10-16 days. The survival rate of oocytes after 10 days of culture (62.8%) was similar to that of oocytes after 12 days of culture (55%) and significantly higher than that of oocytes cultured for 14 and 16 days (52.9 and 24.3%, respectively). No significant difference was observed in the diameter of ooplasm from oocytes cultured for different durations (117.4-118.3 µm). The maturation rates of surviving oocytes after 10 and 16 days of culture (38.3 and 22.7%, respectively) were significantly lower than those of oocytes cultured for 12 and 14 days, and their in vivo counterparts (52.8-62.4%). The number of oocytes with surrounded-nucleolus chromatin was significantly lower in the 10-day culture group (78.4%) as compared with 14-day culture and in vivo counterpart groups (93.6 and 95.1%, respectively). After in vitro maturation and intracytoplasmic sperm injection, no significant difference was observed in the rate of fertilization among oocytes cultured for 12 and 14 days, and their in vivo counterparts (40.5-47.2%). Thus, porcine GOs required at least 12 days to acquire meiotic and fertilization competence, and the culture duration to maximize the number of mature oocytes ranged from 12 to 14 days.

Quantitative and qualitative changes of mitochondria in human preimplantation embryos.

PURPOSE: The oxygen consumption rates (OCRs) in mice and cattle have been reported to change during preimplantation embryogenesis. On the other hand, mitochondrial DNA (mtDNA) copy number has been shown to be unchanged in mice and changed in cattle and pigs. The interactions between mitochondrial functions and mtDNA copy numbers in human embryos during preimplantation development remain obscure. METHODS: Sixteen oocytes and 100 embryos were used to assess mtDNA copy numbers and OCR. Three oocytes and 12 embryos were used to determine cytochrome c oxidase activity. All specimens were obtained between July 2004 and November 2014, and donated from couples after they had given informed consent. Mature oocytes and embryos at 2-14-cell, morula, and blastocyst stages were used to assess their OCR in the presence or absence of mitotoxins. The mtDNA copy number was determined using the samples after analysis of OCR. The relationships between developmental stages and OCR, and developmental stages and mtDNA copy number were analyzed. Furthermore, cytochrome c oxidase activity was determined in oocytes and 4-cell to blastocyst stage embryos. RESULTS: The structure of inner mitochondrial membranes and their respiratory function developed with embryonic growth and the mtDNA copy numbers decreased transiently compared with those of oocytes. The undifferentiated state of inner cell mass cells appears to be associated with a low OCR. On the other hand, the mtDNA copy numbers increased and aerobic metabolism of mitochondria increased in trophectoderm cells. CONCLUSIONS: The mitochondrial respiratory function of human embryos developed along with embryonic growth although the copy numbers of mtDNA decreased transiently before blastulation. OCRs increased toward the morula stage ahead of an increase of mtDNA at the time of blastulation. Data regarding changes in mitochondrial function and mtDNA copy number during preimplantation development of human embryos will be useful for the development of ideal culture media.

Combination of density gradient centrifugation and swim-up methods effectively decreases morphologically abnormal sperms.

Density gradient centrifugation (DGC) and swim-up techniques have been reported for semen preparation in assisted reproductive techniques in humans. We investigated whether semen preparation using a combination of DGC and swim-up techniques could effectively decrease morphologically abnormal human sperms at the ultrastructural level. Semen samples were obtained from 16 infertile males and fractionated by swim-up following DGC. Ultrastructural abnormalities of sperms obtained from original semen, lower layer of swim-up following DGC, and upper layer of swim-up following DGC were analyzed by transmission electron microscopy. The correlation among ultrastructural head abnormality in sperms from the upper layer of swim-up, fertilization in in vitro fertilization, and pregnancy after embryo transfer was also investigated. Furthermore, sperms with DNA fragmentation in the samples processed via a combination of DGC and swim-up was assessed in a sperm chromatin structure assay. Ultrastructural abnormalities in sperm heads and tails in the upper layer after swim-up following DGC was the lowest among the three groups. Sperms with nuclear vacuoles were the most difficult to eliminate using a combination of DGC and swim-up in all types of head abnormalities. A negative correlation was confirmed between the fertilization rates of intracytoplasmic sperm injection and head abnormality of sperms obtained from the upper layer of the swim-up following DGC. Sperms with DNA fragmentation were effectively decreased using the combination of two techniques. In conclusion, the combination of DGC and swim-up effectively decreased the number of sperms with ultrastructural abnormalities both in the head and in the tail. However, sperms with ultrastructural abnormalities that cannot be completely decreased using a combination of DGC and swim-up may impair fertilization in some cases of intracytoplasmic sperm injection.