Clinical outcomes of single blastocyst transfer with machine learning guided noninvasive chromosome screening grading system in infertile patients.

BACKGROUND: Prospective observational studies have demonstrated that the machine learning (ML) -guided noninvasive chromosome screening (NICS) grading system, which we called the noninvasive chromosome screening-artificial intelligence (NICS-AI) grading system, can be used embryo selection. The current prospective interventional clinical study was conducted to investigate whether this NICS-AI grading system can be used as a powerful tool for embryo selection. METHODS: Patients who visited our centre between October 2018 and December 2021 were recruited. Grade A and B embryos with a high probability of euploidy were transferred in the NICS group. The patients in the control group selected the embryos according to the traditional morphological grading. Finally, 90 patients in the NICS group and 161 patients in the control group were compared statistically for their clinical outcomes. RESULTS: In the NICS group, the clinical pregnancy rate (70.0% vs. 54.0%, p < 0.001), the ongoing pregnancy rate (58.9% vs. 44.7%, p = 0.001), and the live birth rate (56.7% vs. 42.9%, p = 0.001) were significantly higher than those of the control group. When the female was ≥ 35 years old, the clinical pregnancy rate (67.7% vs. 32.1%, p < 0.001), ongoing pregnancy rate (56.5% vs. 25.0%, p = 0.001), and live birth rate (54.8% vs. 25.0%, p = 0.001) in the NICS group were significantly higher than those of the control group. Regardless of whether the patients had a previous record of early spontaneous abortion or not, the live birth rate of the NICS group was higher than that of the control group (61.0% vs. 46.9%; 57.9% vs. 34.8%; 33.3% vs. 0%) but the differences were not statistically significant. CONCLUSIONS: NICS-AI was able to improve embryo utilisation rate, and the live birth rate, especially for those ≥ 35 years old, with transfer of Grade A embryos being preferred, followed by Grade B embryos. NICS-AI can be used as an effective tool for embryo selection in the future.

Embryo selection through non-invasive preimplantation genetic testing with cell-free DNA in spent culture media: a protocol for a multicentre, double-blind, randomised controlled trial.

INTRODUCTION: Morphological evaluation is used to select embryos for in vitro fertilisation. However, it does not fully reflect the implantation potential. Preimplantation genetic testing for aneuploidies (PGT-A) can detect embryonic aneuploidy, but biopsy procedure is invasive. Currently, a non-invasive PGT (ni-PGT) approach using spent medium is being evaluated. However, the clinical benefit of ni-PGT has not been clearly demonstrated. A multicentre randomised trial is needed to verify whether ni-PGT can be an new effective tool for evaluating embryos. METHODS AND ANALYSIS: Overall, 1148 couples aged 35~42 (women) receiving in vitro fertilization-intracytoplasmic sperm injection are planned to be enrolled. Couples will be digitally randomised to (1) ni-PGT and (2) conventional morphology groups at a 1:1 treatment ratio. The primary outcome will be the ongoing pregnancy rate related to the first transfer cycle within 6 months after oocyte retrieval. ETHICS AND DISSEMINATION: The study protocol is approved by the Ethics Committee of Peking University Third Hospital and the participating hospitals. The results will be disseminated through international conferences and scientific journals. TRIAL REGISTRATION NUMBER: NCT04339166.

Non-invasive embryo selection strategy for clinical IVF to avoid wastage of potentially competent embryos.

RESEARCH QUESTION: Can a non-invasive embryo transfer strategy provide a reference for embryo selection to be established? DESIGN: Chromosome sequencing of 345 paired blastocyst culture medium and whole blastocyst samples was carried out and a non-invasive embryo grading system was developed based on the random forest machine learning algorithm to predict blastocyst ploidy. The system was validated in 266 patients, and a blinded prospective observational study was conducted to investigate clinical outcomes between machine learning-guided and traditional non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) analyses. Embryos were graded as A, B or C according to their euploidy probability levels predicted by non-invasive chromosomal screening (NICS). RESULTS: Higher live birth rate was observed in A- versus C-grade embryos (50.4% versus 27.1%, P = 0.006) and B- versus C-grade embryos (45.3% versus 27.1%, P = 0.022) and lower miscarriage rate in A- versus C-grade embryos (15.9% versus 33.3%, P = 0.026) and B- versus C-grade embryos (14.3% versus 33.3%, P = 0.021). The embryo utilization rate was significantly higher through the machine learning strategy than the conventional dichotomic judgment of euploidy or aneuploidy in the niPGT-A analysis (78.8% versus 57.9%, P < 0.001). Better outcomes were observed in A- and B-grade embryos versus C-grade embryos and higher embryo utilization rates through the machine learning strategy compared with traditional niPGT-A analysis. CONCLUSION: A machine learning guided embryo grading system can be used to optimize embryo selection and avoid wastage of potential embryos.

Vaginal progesterone gel is non-inferior to intramuscular progesterone in efficacy with acceptable tolerability for luteal phase support: A prospective, randomized, multicenter study in China.

OBJECTIVES: Research suggests the efficacy of progesterone for luteal phase support in assisted reproduction cycles using gonadotropin-releasing hormone analogues. Our study objective was to compare the efficacy of two available preparations of progesterone, vaginal gel and intramuscular injection, for luteal phase support in assisted reproduction cycles. STUDY DESIGN: This study included data gathered from 18 reproductive centers in China. Subjects were randomly allocated to receive progesterone gel or intramuscular progesterone (IMP). The progesterone gel group received micronized progesterone in gel (8%, 90 mg) once daily; the IMP group received IMP (progesterone oil) once daily. The ongoing pregnancy rate was calculated (number of women with a viable pregnancy at 12 weeks divided by the number of women who had undergone an oocyte pickup cycle). RESULTS: A total of 1313 patients were enrolled in the study, 1248 of whom began treatment. The intention-to-treat set included 527 and 531 patients in the gel and IMP groups, respectively. The ongoing pregnancy rate in the progesterone gel group was non-inferior to that in the IMP group (48.4% [95% confidence interval (CI): 44.0, 52.8] vs. 46.3% [95% CI: 42.0, 50.7]); the between-group rate difference was 2.1% (-4.0, 8.1). There was no difference between the gel group and IMP group on most secondary endpoints, including implantation rate, biochemical pregnancy rate, clinical pregnancy rate, multiple pregnancy rate, early abortion rate, and vaginal bleeding rate, but there was a between-group difference in luteal phase bleeding rate. The safety analysis showed no difference in the incidence of total adverse events. CONCLUSIONS: Progesterone gel showed good efficacy and safety outcomes and therefore provides an alternative method of luteal support in Chinese in vitro fertilization patients.

Chromosome screening using culture medium of embryos fertilised in vitro: a pilot clinical study.

BACKGROUND: Previous studies from this as well as other research groups suggested that non-invasive chromosome screening (NICS) with embryo culture medium can be used to identify chromosomal ploidy and chromosomal abnormalities. We here report a series of clinical cases utilizing the technology. METHODS: A total of 45 couples underwent in vitro fertilisation during a period between February 2016 and February 2017. Karyotyping revealed normal chromosomes in both partners in 23 couples, and chromosomal rearrangements in at least one partner in 22 couples. Intracytoplasmic sperm injection (ICSI) was used for fertilization. NICS was carried out using embryo culture medium at the blastocyst stage via multiple annealing and looping-based amplification cycles, whole-genome amplification and next-generation sequencing. RESULTS: A total of 413 embryos were obtained; 170 blastocysts were subjected to NICS. The screening showed euploidy in 79 embryos, aneuploidy in 52 embryos, and mosaic ploidy for 33 embryos. The rate of euploidy was comparable in couples with normal karyotype (50.7%; 38/75) vs. chromosomal rearrangement (43.2%; 41/95). A total of 52 euploid embryos (50 oocyte retrieval cycles) were transferred in 43 women. Biochemical pregnancy rate was 72.0% (36/50). Clinical pregnancy rate was 58.0% (29/50). The rate of spontaneous miscarriage was 3/29 (none with chromosomal aneuploidy). A total of 27 healthy babies were delivered. CONCLUSIONS: NICS could identify embryo chromosomal abnormalities in couples either with or without chromosomal rearrangement, with satisfying clinical outcomes.

Presence of human herpes virus 1-thymidine kinase in testis of azoospermic infertile herpes-infected patients.

Infection with human herpes virus 1 (HHV1) is a suspected cause of human male infertility. However, the correlation between HHV1 infection and infertility is still unclear. We have previously generated transgenic rats that ectopically express the HHV1 thymidine kinase gene (HHV1-TK) in post-meiotic spermatids and found they had aberrant spermatogenesis and infertility. Therefore, we hypothesized that human infertility might be caused by HHV1 infection. Here, we examined whether HHV1-TK is expressed in human testis by analyzing the presence of its transcript and protein. Specimens were collected by biopsy from 30 azoospermic infertile male patients. RT-PCR and immunohistochemistry showed that 23 patients were positive for HHV1-TK expression, while seven patients were negative. Thus, we demonstrated HHV1-TK expression, indicating HHV1 infection, in the testis of human azoospermic infertile males for the first time; our findings represent a great advancement toward the verification of our hypothesis that HHV1-TK expression might cause human infertility.

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.

Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

The effect of endometrial thickness on the day of hCG administration on pregnancy outcome in the first fresh IVF/ICSI cycle.

The aim of this study was to investigate the effect of endometrial thickness (EMT) on human chorionic gonadotropin (hCG) day on in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcome. A retrospective study was conducted on the clinical data of 756 patients in their first fresh IVF/ICSI cycle at the Wuxi Maternity and Child Health Hospital. Compared with the pregnancy failure group, the clinical pregnancy group had more transferable embryos and good-quality embryos and had a thicker endometrium (p < 0.05). The endometrial pattern was not significantly different between the two groups. EMT was found to be an independent prognostic factor for clinical pregnancy (adjusted OR = 1.25, 95% CI: 1.15-1.36, p < 0.01). Seven hundred and fifty-six cycles were categorized into three groups upon EMT on the hCG day: group 1 (EMT < 8 mm), 2 (EMT 8-14 mm) and 3 (EMT > 14 mm). Group1 had significantly lower clinical pregnancy, embryo implantation and live birth rates compared with group 2 and 3 (p < 0.01), while there was no significant difference in either spontaneous abortion or multiple-birth rate among these three groups. It was concluded that EMT on the hCG day was associated with pregnancy outcome in the first fresh IVF/ICSI cycle. A higher clinical pregnancy rate could be achieved when EMT ≥ 8 mm, and no adverse pregnancy outcome was observed when EMT > 14 mm.

Vitamin D Status among Young Children Aged 1-3 Years: A Cross-Sectional Study in Wuxi, China.

BACKGROUND: The increasingly recognized importance of vitamin D has been discussed and vitamin D status among young children has attracted widespread attention in recent years. However, study on vitamin D status in young children aged 1-3 y is limited in China. OBJECTIVE: To evaluate the nutritional vitamin D status of young children aged 1-3 y in Wuxi, southeastern China. METHODS: A large cohort of 5,571 young children aged 1-3 y were recruited in this study who visited the child health clinics at the Wuxi Maternity and Child Health Hospital (latitude 31.57°N) during January 2014 to January 2015. Wuxi was located in southeastern China at a latitude of 31.57°N. Finger-stick blood sampling was conducted in all the subjects and serum 25-Hydroxyvitamin D (25(OH)D) levels were measured to evaluate their vitamin D status. RESULTS: In this study, serum 25(OH)D levels of young children at the age of 1-3 years ranged from 20.6-132.9 nmol/L (Median: 71.5 nmol/L). 16.1% of the population had vitamin D deficiency (<50 nmol/L), while 38.8% of the subjects had a sufficient (50-74.9 nmol/L) vitamin D level. An optimal vitamin D status (≥75 nmol/L) was found in 45.1% of the young children. The prevalence of vitamin D deficiency was higher in autumn (19.5%) than in summer (12.1%). There was no significant difference in vitamin D status between genders. The binary logistic regression analysis revealed that child age was strongly associated with vitamin D deficiency (adjusted OR: 1.173; 95%CI: 1.053-1.308; P = 0.004). CONCLUSIONS: The prevalence of vitamin D deficiency was 16.1% among young children aged 1-3 y in Wuxi. Season and child age were associated with their vitamin D status. It is implied that young children should receive adequate amounts of vitamin D supplementation and spend more time outdoors to prolong the sunlight exposure when they grow older.

Toll-like receptor 4 polymorphisms to determine acute pancreatitis susceptibility and severity: a meta-analysis.

AIM: To investigate the correlation of toll-like receptor 4 (TLR4) gene Asp299Gly and Thr399Ile polymorphisms and acute pancreatitis (AP) risk and severity. METHODS: To get a more precise estimation of the relationship, a comprehensive search was performed to examine all the eligible studies of TLR4 Asp299Gly and Thr399Ile polymorphisms and AP risk. The odds ratios with 95% confidence intervals were used to assess the strength of the association. Publication bias was analyzed by Begg's funnel plots. RESULTS: In total, six studies with 1255 cases and 998 controls were included in this meta-analysis. Totally, no significant associations were found between TLR4 Asp299Gly or Thr399Ile polymorphisms and AP risk using five models with high homogeneity (P > 0.05). Furthermore, stratification analysis by ethnicity or assay also found no significant association in these two polymorphisms (P > 0.05), and TLR4 Asp299Gly was not associated with AP severity (P > 0.05). In addition, no publication bias was found in these studies (P > 0.05). CONCLUSION: Our current meta-analysis suggests that TLR4 Asp299Gly and Thr399Ile polymorphisms may not be risk factors to AP susceptibility.

Roles of PPARγ/NF-κB signaling pathway in the pathogenesis of intrahepatic cholestasis of pregnancy.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is the most prevalent pregnancy specific liver disease. However, the pathogenesis and etiology of ICP is poorly understood. AIM: To assess the expression of peroxisome proliferator-activated receptorγ (PPARγ) and nuclear factor kappa B (NF-κB) in placenta and HTR-8/SVneo cell, and evaluate the serum levels of cytokines, bile acids, hepatic function and lipids in control and ICP patients and the fetal outcome, in order to explore the role of PPARγ/NF-κB signaling pathway in the possible mechanism of ICP. METHODS: Clinical data of the pregnant women were collected and serum levels of cytokines, bile acids, hepatic function and lipids were measured. Expressions of PPARγ and NF-κB in placenta and HTR-8/SVneo cell were determined. The new-born information was collected to demonstrate the relationship between PPARγ/NF-κB signaling pathway and ICP. RESULTS: The serum levels of bile acids, hepatic function, triglycerides (TG), total cholesterol (TC), IL-6, IL-12 and TNF-α in ICP group were significantly increased (P<0.01), and serum level of IL-4 was significantly decreased (P<0.01). PPARγ and NF-κB staining were found in the membrane and cytoplasm of placental trophoblast cell. The expression of PPARγ and NF-κB were significantly higher in ICP group and taurocholate acid (TCA) treated HTR-8/SVneo cell (P<0.01). The new-born information in severe ICP group were significantly different as compared to that in control group (P<0.05), and part of information in mild ICP group were also difference to that in control group (P<0.05). CONCLUSIONS: The higher expressions of PPARγ and NF-κB in ICP placenta and TCA treated HTR-8/SVneo cell, together with the abnormal serum levels of cytokines, might induced by the imbalance of inflammatory and immune reaction, and then disturb placental bile acid and serum lipids transportation, finally result in fatal cholestasis which probably be one of the mechanism of ICP.

Alterations in the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology.

STUDY QUESTION: How does the frequency of trinucleotide repeat dynamic mutations in offspring conceived through assisted reproductive technology (ART) compare with the frequency of these mutations in control offspring conceived from spontaneous pregnancies? SUMMARY ANSWER: There is a slight increase in dynamic mutation instability in offspring conceived through ART compared with the naturally conceived offspring. WHAT IS KNOWN ALREADY: There is evidence to suggest that ART can increase the risk of birth defects and karyotypic abnormalities. However, the accumulating evidence of an association between ART and de novo genetic aberrations is controversial. STUDY DESIGN, SIZE, DURATION: A prospective clinical observational study was performed on 246 families recruited from an in vitro fertilisation (IVF) centre at a tertiary-care, university-affiliated teaching hospital from 2008 to 2012. The study included 147 ART families [75 IVF and 72 intracytoplasmic sperm injection (ICSI)] in the study group and 99 natural-conception families in the control group. PARTICIPANTS, SETTING, METHODS: Parental, umbilical cord and infant peripheral blood samples were collected, and the trinucleotide repeats of the ATN1, AR, ATXN1, ATXN3, Huntington, DMPK and FMR-1 genes were investigated between the generations; these genes were chosen due to their ability to undergo dynamic mutation. The frequencies and sizes of the mutational repeats, as well as the intergenerational instability, were measured. MAIN RESULTS AND THE ROLE OF CHANCE: In 2466 transmissions identified in the ART offspring, 2.11% (n = 52/2466) of the alleles were unstable upon transmission, while in the control group offspring, the frequency of dynamic mutation was 0.77% (n = 10/1300); this difference was statistically significant (P < 0.01). The unstable transmission alleles were detected in 32 (2.48%) of the 1288 alleles from the IVF offspring and in 20 (1.70%) of the 1178 alleles from the ICSI offspring; both of these frequencies were significantly different from that of naturally conceived offspring (0.77%) (P < 0.01 and P < 0.05, respectively). However, there were no significant differences in the sizes of the mutational repeats or in the rates of expansion or contraction among the three groups (P > 0.05). The repeat copy numbers of the examined genes were found to be within the normal ranges in all parents and infants. LIMITATIONS, REASONS FOR CAUTION: One strength of our study is the relatively large sample size; we were able to detect mutations in seven common dynamic genes, and this large sample size allowed us to detect unstable alleles. Although we observed a clear alteration in the frequency of dynamic mutation in the ART offspring compared with controls, further studies are urgently needed to confirm this observation and determine the cause of this phenomenon. WIDER IMPLICATIONS OF THE FINDINGS: DNA microsatellite analysis provides an important tool to assess genomic instability. In this study, we report an association between ART and the frequency of dynamic mutation. The instability could be a reflection of the core infertility problem, the controlled ovarian hyperstimulation and/or the in vitro culture conditions.

Characterization of a pituitary-tumor-derived cell line, TtT/GF, that expresses Hoechst efflux ABC transporter subfamily G2 and stem cell antigen 1.

The anterior lobe of the pituitary gland is composed of five types of endocrine cells and of non-endocrine folliculo-stellate cells that produce various local signaling molecules. The TtT/GF cell line is derived from pituitary tumors, produces no hormones and has folliculo-stellate cell-like characteristics. The biological function of TtT/GF cells remains elusive but several properties have been postulated (support of endocrine cells, control of cell proliferation, scavenger function). Recently, we observed that TtT/GF cells have high resistance to the antibiotic G418 and low influx for Hoechst 33342, indicating the presence of ATP-binding cassette (ABC) transporters that efflux multiple drugs, i.e., a property similar to that of stem/progenitor cells. Therefore, we examine TtT/GF cells for the presence of ABC transporters, for the efflux ability of Hoechst 33342 and for those genes characteristic of TtT/GF cells. Real-time polymerase chain reaction (PCR) for ABC transporters demonstrated that Abcb1a, Abcb1b and Abcg2, regarded as stem cell markers, were characteristically expressed in TtT/GF cells but not in Tpit/F1 and LßT2 cells. Furthermore, the remarkable low-efflux ability of Hoechst 33342 from TtT/GF cells was confirmed by using inhibitors and contrasted with the abilities of Tpit/F1 and LßT2 cells. The high and specific expression of stem cell antigen 1 (Sca1) in TtT/GF cells was confirmed by real-time PCR. We also demonstrated those genes that are expressed abundantly and characteristically in TtT/GF, suggesting that TtT/GF cells have unique characteristics similar to those of stem/progenitor cells of endothelial or mesenchymal origin. Thus, the present study has revealed an intriguing property of TtT/GF cells, providing a new clue for an understanding of the function of this cell line.

Detection of human herpesviruses (HHVs) in semen of human male infertile patients.

Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21-49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.

[A new puncture needle with multiple holes for testicular sperm extraction].

OBJECTIVE: To evaluate a new puncture needle with multiple holes (National Invention Patent of China: ZL 2010202466554) in testicular sperm extraction for infertile males. METHODS: This study included 215 azoospermia patients, who underwent testicular sperm extraction with a new puncture needle with multiple holes (group A, n = 133), by open biopsy (group B, n = 37), or with a fine needle (group C, n = 45). RESULTS: The first-time success rate was 100% in group A, 19% in B and 100% in C. The average operation time was obviously shorter in group A ([3 +/- 1] min) than in B ([15 +/- 3] min) and C ([7 +/- 2] min). The rate of postoperative complications was 3.0% in group A, significantly lower than in B (21.6%) and C (11.1%). CONCLUSION: The new puncture needle with multiple holes, with its advantages of accuracy, high first-time success rate, minimal invasiveness and low rate of complications, deserves to be generally applied in testicular sperm extraction.

Accumulated HSV1-TK proteins interfere with spermatogenesis through a disruption of the integrity of Sertoli-germ cell junctions.

Transgenic rats show spermatid-specific ectopic expression of the reporter gene, herpes simplex virus type1 thymidine kinase (HSV1-TK), in the testes and have demonstrated male infertility. However, the disruption of spermatogenesis and the underlying molecular mechanisms in these transgenic animals have not been well clarified. In this study, light and electron microscopic observations were performed to characterize the morphological changes in the testes. To explore the molecular mechanisms of male infertility in the HSV1-TK transgenic rat, cDNA microarray and quantitative real-time PCR analyses were performed. The seminiferous tubules of 3-month-old transgenic rats showed morphological alterations including seminiferous epithelial sloughing, vacuolization, and degeneration of spermatogenic cells, suggesting a failure of Sertoli-germ cell interaction. Components of the epididymal lumen from transgenic rats included abnormal spermatozoa, degenerating round spermatids and abnormal elongated spermatids indicating an appearance of direct impairment of spermiogenesis. cDNA microarray and real-time PCRanalyses revealed significant changes (P<0.05) in the gene expression level in six genes, testin, versican, mamdc1, fgf7, ostf1 and cnot7. Among them, testin drew most of our attention, since the testin gene is a sensitive marker for disruption of Sertoli-germ cell adhesion. Thus, our results suggest that the accumulation of HSV1-TK in the spermatids not only directly interferes with spermiogenesis but also disrupts spermatogenesis through a disruption of Sertoli-germ cell adhesions. It is important to explore the testicular actions of the HSV1-TK protein in transgenic experimental models and thereby gain clues to find an appropriate treatment for HSV-infected patients exhibiting human male infertility, as has been recently observed.

Lack of an association human dioxin detoxification gene polymorphisms with endometriosis in Japanese women: results of a pilot study.

OBJECTIVES: Endometriosis is a chronic disease caused by the presence of endometrial tissue in ectopic locations outside the uterus. Chronic exposure to the environmental pollutant dioxin has been correlated with an increased incidence in the development of endometriosis in non-human primates. We have therefore examined whether there is an association between the polymorphisms of ten dioxin detoxification genes and endometriosis in Japanese women. METHODS: This was a pilot study in which 100 patients with endometriosis and 143 controls were enrolled. The prevalence of five microsatellite and 28 single nucleotide polymorphism markers within ten dioxin detoxification genes (AhR, AHRR, ARNT, CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, GSTT1, NAT2) was examined. RESULTS: Taking into account that this analysis was a preliminary study due to its small sample size and genetic power, the results did not show any statistically significant difference between the cases and controls for any of the allele and genotype frequency distributions examined. In addition, no significant associations between the allele/genotype of all polymorphisms and the stage (I-II or III-IV) of endometriosis were observed. CONCLUSION: Based on the findings of this pilot study, we conclude the polymorphisms of the ten dioxin detoxification genes analyzed did not contribute to the etiology of endometriosis among our patients.

Failure to detect significant association between estrogen receptor-alpha gene polymorphisms and endometriosis in Japanese women.

OBJECTIVES: The aim of the study was to test whether estrogen receptor 1 (ESR1) gene polymorphisms are correlated with the risk of the development of endometriosis in Japanese women, as a preliminary study. METHODS: To compare allelic frequencies and genotype distributions, a case-control study of 100 affected women and 143 women with no evidence of disease was performed using 10 microsatellite repeat markers and 66 single-nucleotide polymorphisms (SNPs) in the ESR1 gene region. RESULTS: Although our results might be insufficient to detect genetic susceptibility, owing to the small sample size and low genetic power, statistical analysis of the differences in allelic frequency between the cases and controls at each microsatellite locus demonstrated that no microsatellite locus in the ESR1 gene displayed a significant association with the disease when multiple testing was taken into account. Also, there were no statistically significant differences in the SNP allele frequencies and genotypes between the cases and controls when multiple testing was taken into account. CONCLUSION: The findings in our pilot study suggest that ESR1 polymorphisms do not contribute to endometriosis susceptibility.

Does aberrant DNA methylation occur in human uterine leiomyomas? An attempt of genome-wide screening by MS-RDA.

OBJECTIVE: Uterine leiomyoma are very common benign tumors in women of reproductive age. However, the molecular mechanisms of cause and development of these tumors are poorly understood. This study attempts to examine whether or not aberrant DNA methylation occurred in these tumors. METHODS: We carried out a genome-wide screen for aberrant DNA methylation, adopting methylation-sensitive-representational difference analysis (MS-RDA) using normal adjacent myometria as tester and myoma tissue driver. CONCLUSION: A total of 192 clones identified by MS-RDA were sequenced, 27 DNA fragments derived from CpG islands (CGIs) were isolated, and seven of them were from CGI in the 5' regions of known genes, which include CHARC1, FAM44B, FLJ33655, HSUP, MLLT3, SLC16A1, and ZNF96. Then, methylation statuses of those CGIs were analyzed by methylation-specific polymerase chain reaction using 5 primary samples of human uterine leiomyoma. Aberrant DNA methylation did not observed in 7 genes in 5 human uterine leiomyoma eventually. This study is insufficient to identify aberrant DNA methylation occurring in the human uterine leiomyoma, a large population of primary samples and more attempts, such as the use of cell lines or primary monolayer cultures established from tissue samples, are warranted to clarify this issue.

Placental and maternal serum inhibin A in patients with preeclampsia and small-for-gestational-age.

AIM: The purpose of this study was to determine the levels of inhibin A simultaneously in the maternal serum and placental extract in preeclampsia (PE) with or without small-for-gestational-age (SGA) and normal controls at term, and to evaluate the relationship among changes in serum and placental inhibin A according to the severity of PE and PE with or without SGA. MATERIAL AND METHODS: This study involved 40 pregnant women; normal (n = 20), and PE (n = 20), the latter of who were classified into (i) mild (n = 10) and severe PE (n = 10); (ii) PE with SGA (n = 7) and without SGA (n = 13). Inhibin A concentrations were quantified by enzyme-linked immunosorbent assay (ELISA) in the maternal serum and placental extract. Inhibin-α subunit in the placenta was stained by immunohistochemistry (IHC), and its intensity was graded by a semiquantitative scoring method. RESULTS: There was a positive correlation in inhibin A concentrations between the serum and placental extract (r = 0.57, P < 0.001). Both maternal serum and placental inhibin A in PE groups were significantly higher than in controls, but there was no severity-dependent increase of inhibin A when compared with mild and severe PE. There was no difference in inhibin A levels between PE with and without SGA. Moreover, the inhibin-α subunit was predominantly abundant in the cytoplasm of the syncytiotrophoblasts, where the PE groups showed higher staining intensity than the controls (P < 0.000). CONCLUSION: Serum inhibin A level might be a useful biomarker for diagnosis and monitoring of PE.