Direct conversion from long-acting testosterone replacement therapy to Natesto allows for spermatogenesis resumption: Proof of concept.

Long-acting testosterone replacement therapy (TRT) suppresses spermatogenesis. A short-acting TRT, Natesto, maintains spermatogenesis in some men. This study evaluated hormonal and semen parameters converting men from long-acting TRT to Natesto. Baseline hormones, again on long-acting TRT and 1 month after converting to Natesto, as well as semen parameters 3 months after converting to Natesto were assessed. Twenty-seven men were directly converted from long-acting forms of TRT to Natesto. Mean duration on long-acting TRT was 24.3 ± 19 months. Testosterone levels were similar on long-acting forms of TRT and Natesto, however; E2 levels were significantly lower on Natesto. Ten men had semen analyses demonstrating azoospermia while on long-acting TRT, the remainder were presumed to be azoospermic or severely oligospermic which has been well established as an effect of long-acting TRT. All 27 men had resumption of spermatogenesis with a mean sperm concentration of 50.7 million/ml after converting to Natesto, considered within the fertile range. One couple achieved a pregnancy 4 months after converting to Natesto. Hypogonadal men on long-acting TRT interested in resumption of spermatogenesis may convert directly to Natesto for an opportunity to do so while remaining on a form of TRT and achieving lower E2 levels.

The expression of pregnancy-associated plasma protein-A (PAPP-A) in human blastocoel fluid-conditioned media: a proof of concept study.

PURPOSE: The aim of this study was to determine if pregnancy-associated plasma protein-A (PAPP-A), typically measured in maternal serum and a potential predictor of adverse maternal and fetal outcomes such as spontaneous miscarriage, pre-eclampsia, and stillbirth, is expressed in blastocoel fluid-conditioned media (BFCM) at the embryonic blastocyst stage. DESIGN: This is an in vitro study. METHODS: BFCM samples from trophectoderm-tested euploid blastocysts (n = 80) from in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) patients were analyzed for PAPP-A mRNA. BFCM was obtained from blastocyst stage embryos in 20 uL drops. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy prior to blastocyst vitrification and BFCM collection for snap freezing. cfDNA was synthesized using BFCM collected from 80 individual euploid blastocysts. Next, real-time qPCR was performed to detect expression of PAPP-A with GAPDH for normalization of expression in each sample. RESULTS: PAPP-A mRNA was detected in 45 of 80 BFCM samples (56.3%), with varying levels of expression across samples. CONCLUSION: Our study demonstrates the expression of PAPP-A in BFCM. To our knowledge, this is the first study to report detection of PAPP-A mRNA in BFCM. Further studies are required and underway to investigate a greater number of BFCM samples as well as the possible correlation of PAPP-A expression with pregnancy outcomes of transferred euploid blastocysts. If found to predict IVF and obstetric outcomes, PAPP-A may provide additional information along with embryonic euploidy for the selection of the optimal blastocyst for embryo transfer.

The effects of hysteroscopic morcellation of endometrial polyps on frozen embryo transfer outcomes.

OBJECTIVE(S): To determine if hysteroscopic removal of endometrial polyps, specifically via morcellation of polyps, affects implantation rate (IR), clinical pregnancy rate (CPR), spontaneous abortion (SAB) rate, and live birth rate (LBR) in first frozen embryo transfer (FET) cycles. STUDY DESIGN: Retrospective chart review, with data abstracted from the charts of all first autologous oocyte frozen embryo transfer (FET) cases (n = 135) at a single fertility center from January 2018 through June 2020. Subjects were grouped into (A) hysteroscopic polypectomy prior to first FET (n = 25) or (B) no hysteroscopic polypectomy prior to first FET (n = 110). The primary outcome was live birth rate (LBR). Secondary outcomes were implantation rate (IR), clinical pregnancy rate (CPR), and spontaneous abortion (SAB) rate. RESULTS: We found no difference between the groups in terms of the primary outcome (LBR) or the secondary outcomes IR, CPR, and SAB rate. CONCLUSION(S): The data analyzed here suggest that hysteroscopic morcellation of endometrial polyps has no adverse effect on IR, SAB rate, CPR, or LBR among first FET cases after this type of polypectomy.

The impact of ipsilateral testicular atrophy on semen quality and sperm DNA fragmentation response to varicocele repair.

Varicoceles adversely impact semen quality and sperm DNA fragmentation, which typically improve with surgical repair. Some men with varicoceles have ipsilateral testicular atrophy due to damage from the varicocele. This study assessed semen quality and the sperm DNA fragmentation index (DFI) response to varicocele repair in men with ipsilateral testicular atrophy (TA) versus men with no testicular atrophy (NTA). Semen parameter values and DFI in both groups were compared preoperatively and postoperatively. The Mann-Whitney U test and the Wilcoxon signed-rank test were used where appropriate. There were 20 men in the TA group and 121 men in the NTA group with no difference in age, varicocele grade, or preoperative semen parameter values between the two groups. The NTA group had a higher preoperative DFI than the TA group. Both groups showed improvement in semen quality postoperatively, only the TA group showed a significant improvement in DFI, whereas the NTA group showed significant improvements in several parameter values and DFI. The change from preoperative to postoperative parameter values when comparing the two groups revealed a difference in total sperm motile count and DFI, with a larger mean improvement in the NTA group than in the TA group. Both TA and NTA groups showed improved semen quality and DFI after varicocele repair, but the NTA group had more improvement than the TA group. However, only total motile count (TMC) and DFI had a significantly greater mean change in preoperative to postoperative response in the NTA group than in the TA group.

Converting Men From Clomiphene Citrate to Natesto for Hypogonadism Improves Libido, Maintains Semen Parameters, and Reduces Estradiol.

OBJECTIVE: To evaluate outcomes including libido, semen parameters, testosterone, estradiol (E2), follicle stimulating hormone (FSH), and luteinizing hormone when converting men with low libido on Clomiphene Citrate (CC) to Natesto. METHODS: A retrospective chart review was performed. Baseline hormones prior to treatment, and again on CC and Natesto, as well as semen parameters on CC and on Natesto were assessed. RESULTS: In 41 men, there was no difference in serum testosterone levels on CC vs Natesto, however; there was a significantly higher E2 on CC than on Natesto. Although FSH levels were significantly lower on Natesto than at baseline, the mean FSH level on Natesto remained in the normal reference range. There was no difference in luteinizing hormone levels at baseline vs on Natesto. There was not a significant difference in semen parameter values when men were on CC vs when they were on Natesto for 3 months. At 3 months after changing to Natesto, 38 of 41 (92.7%) men reported significantly improved libido on Natesto when compared to CC. CONCLUSION: Men on CC and Natesto reach eugonadal testosterone levels, however; on CC the E2 level nearly doubled from baseline, and converting men from CC to Natesto returned E2 to nearly baseline levels. There was not a detrimental effect on semen parameters, and there was subjective reporting of improved libido after converting from CC to Natesto in this cohort, but further long-term studies are needed prior to Natesto being established as a definitive treatment for hypogonadism for men desiring to maintain fertility.

A comprehensive assessment of predictors of fertility outcomes in men with non-obstructive azoospermia undergoing microdissection testicular sperm extraction.

BACKGROUND: Microdissection testicular sperm extraction (microTESE) in men with non-obstructive azoospermia (NOA) is the procedure that results in the highest number of sperm cells retrieved for in vitro fertilization (IVF). This study presents a novel assessment of predictors of sperm retrieval as well as downstream embryology and pregnancy outcomes in cases of men with NOA undergoing microTESE. METHODS: A retrospective chart review of 72 men who underwent microTESE for predictors of fertility outcomes including sperm retrieved at microTESE, embryology progression to embryo transfer (ET), clinical pregnancy, live birth, and surplus sperm retrieved for additional IVF/intracytoplasmic injection cycles beyond one initial cycle. Statistical models for each of these outcomes were fitted, with a p-value of < 0.05 considered significant for the parameters estimated in each model. RESULTS: Seventy-two men underwent microTESE, and 51/72 (70.8%) had sperm retrieved. Of those, 29/43 (67.4%) reached ET. Of the couples who underwent ET, 21/29 (72.4%) achieved pregnancy and 18/29 (62.1%) resulted in live birth. Of the men with sperm retrieved, 38/51 (74.5%) had surplus sperm cryopreserved beyond the initial IVF cycle. Age, testicular volume, FSH, and testicular histopathology were assessed as predictors for sperm retrieved at microTESE, progression to ET, pregnancy, live birth, and surplus sperm. There were no preoperative predictors of sperm retrieval, clinical pregnancy, or live birth. Age predicted reaching ET, with older men having increased odds. FSH level had a negative relationship with surplus sperm retrieved. Men with hypospermatogenesis histology had higher rates of sperm retrieval, clinical pregnancy, live birth, and having surplus sperm. CONCLUSIONS: Men who underwent microTESE with a hypospermatogenesis histopathology had better outcomes, including higher rates of sperm retrieval, clinical pregnancy, live birth, and having surplus sperm retrieved. Increasing male partner age increased the odds of reaching ET. No other clinical factors were predictive for the outcomes considered.

Factors associated with subchorionic hematoma formation in pregnancies achieved via assisted reproductive technologies.

PURPOSE: To determine if certain clinical and/or embryologic factors are independently associated with the increased prevalence of subchorionic hematoma (SCH) among pregnancies achieved via in vitro fertilization (IVF) with fresh embryo transfer (ET). DESIGN: Retrospective chart review. METHODS: In this retrospective study, data were abstracted from 210 autologous oocyte IVF clinical pregnancies that resulted from fresh ET at a single fertility center from January 2012 through December 2016. Clinical and embryology laboratory variables were analyzed as possible factors associated with the presence or absence of SCH in IVF pregnancies via bivariate associations and multivariable logistic regression analyses. Independent variables included prior uterine surgery versus no uterine surgery, peak estradiol, and progesterone levels, day 3 (n = 92) versus day 5 (n = 118) ET, and assisted hatching versus no assisted hatching. Among the day 5 ET subgroup of 118 patients, 117 had data for the variables inner cell mass (ICM) grading and trophectoderm (TE) because one day 5 ET was at the morula stage. RESULTS: We found a significant bivariate association between TE grading and SCH, where cases with TE grade "A" were significantly less likely to have SCH compared with cases with grades "B" or "C." This significant difference remained when adjusting for the other factors considered in a multivariable logistic regression model for the probability of SCH. CONCLUSIONS: The data analyzed here suggest that a less-advanced trophectoderm grade may be a potential factor that is associated with the presence of SCH in pregnancies achieved via IVF.

Sertoli cell only syndrome induced by a varicocele.

A case is reported which describes the severity of testicular histological damage that can be induced by a high-grade varicocele in a male with secondary infertility. A chart review of a patient's case was performed. A 34-year-old male with a three-and-a-half-year-old son who was conceived spontaneously with timed intercourse, with a grade three left varicocele, who's semen parameters progressed to non-obstructive azoospermia (NOA). He did not regain sperm in the ejaculate three or six months post left subinguinal microsurgical varicocele repair. He underwent bilateral microdissection testicular sperm extraction (microTESE) without identification of sperm in the testicular samples. A testicular biopsy from the time of microTESE revealed a Sertoli cell only pattern. A high-grade varicocele has the potential to induce sufficient testicular damage to result in the most severe testicular histological architecture associated with non-obstructive azoospermia (NOA), Sertoli cell only syndrome (SCOS).

Serum Antimüllerian hormone does not predict elevated progesterone levels among women who undergo controlled ovarian hyperstimulation for in vitro fertilization.

Serum Antimüllerian hormone (AMH) has been shown to predict various in vitro fertilization (IVF) outcomes. AMH and progesterone (P) are products of granulosa cells of the ovary. Since overall granulosa cell number directly correlates with oocyte number and AMH production, the aim of this study is to evaluate whether or not serum AMH is associated with elevated P during controlled ovarian hyperstimulation (COH) for IVF. For this retrospective study, data were abstracted from charts of first IVF cycles of women (n = 201) who had undergone COH between May 2014 and May 2017. Groups were as follows: (A) AMH < 1 ng/mL (n = 32), (B) AMH 1-3.99 ng/mL (n = 109), (C), AMH ≥ 4 ng/mL (n = 60). The primary outcome measure was serum P level at trigger prior to oocyte retrieval. Mean serum P levels among groups A, B, and C were 0.92 ng/mL, 0.96 ng/mL, and 0.84 ng/mL, respectively. One-way ANOVA showed that there was no difference in mean serum P level among groups A, B, and C (p-value = 0.28). Multivariable linear regression with P as the dependent variable showed that total gonadotropin dose and peak estradiol level on day of trigger each had a significant positive relationship with P, and clinical pregnancy had a significant negative relationship. Although AMH is a predictor of certain IVF outcomes, AMH is not a predictor of elevated serum P level at trigger among women who undergo COH for IVF.

Ibuprofen does not have an adverse impact on semen parameters.

PURPOSE: A recent study suggested that ibuprofen may alter testicular physiology in a state of compensated hypogonadism, but only evaluated spermatogenic cells in a laboratory ex-vivo model with no significant effect, and found no significant change in follicle stimulating hormone (FSH) in men treated with ibuprofen. The study did not evaluate the impact of ibuprofen use on clinical semen parameters, which has not been assessed to date. The purpose of this study was to evaluate the impact of ibuprofen on semen parameters. METHODS: In a retrospective chart review from October 2012 to February 2018, 64 men had semen analyses revealing leukocytospermia and were treated with a 3-week course of ibuprofen 600 mg every 8 hours (1800 mg per day) and had a repeat semen analyses 3 weeks later. RESULTS: Of the 64 men diagnosed with leukocytospermia, 51 returned for post-treatment semen analyses. Parameters included semen volume, sperm concentration, motility, TMC, and forward progression. Morphology was excluded as it could not be standardized between assessments with strict Kruger criteria versus WHO fourth edition criteria depending on the lab in which it was performed. The mean age of these men was 35 (SD 4.6). There was no difference in mean abstinence intervals prior to semen analyses for the pre-treatment and post-treatment data. There was no significant difference in pre-treatment and post-treatment semen volumes, sperm concentrations, motility, TMC, or forward progression. CONCLUSIONS: Among men with leukocytospermia, the treatment with a 3-week course of ibuprofen at 1800 mg per day did not demonstrate a significant adverse impact on semen volume, sperm concentration, motility, TMC, or forward progressive motility when compared to pre-treatment semen analyses parameters.

Promescent Has a Cytotoxic Impact on Fresh Human Sperm In Vitro.

OBJECTIVE: To assess the impact of Promescent, a commonly used topical medication for premature ejaculation (PE), on human sperm motility, forward progression (FP), viability, and sperm DNA fragmentation (SDF) in vitro. MATERIALS AND METHODS: Aliquots from specimens for diagnostic semen analyses from patients (n = 20) presenting to a couple's fertility center andrology laboratory for fertility testing were included after the full diagnostic semen analyses were performed. Samples that met the World Health Organization's fifth edition criteria as "normal" had Promescent applied to an aliquot of the sample and motility, FP, viability, and SDF were compared with an aliquot that was not treated with Promescent. Institutional review board exemption was obtained. Statistical analysis was performed by Student t test with a P value of <.05 considered as statistically significant. RESULTS: Promescent had a cytotoxic effect on sperm, which resulted in a statistically significant decrease in mean motility, FP, and viability as compared with corresponding control group samples, which did not have Promescent applied. There was no statistically significant difference in SDF between the 2 groups. CONCLUSION: PE is estimated to affect up to 39% of men and is one of the most common self-reported male sexual disorders. There is an overlap among men with PE and those trying to achieve a pregnancy, and Promescent is a commonly used topical treatment for PE. Although there was no difference in SDF between the 2 groups, Promescent had a cytotoxic impact on sperm.

Imipenem heteroresistance induced by imipenem in multidrug-resistant Acinetobacter baumannii: mechanism and clinical implications.

Acinetobacter baumannii has emerged as a major pathogen causing nosocomial infections, particularly in critical patients admitted to the Intensive Care Unit. Increasing resistance to carbapenems in A. baumannii has been observed worldwide. Here we report the clinical impact and mechanism of imipenem heteroresistance (imipenem minimum inhibitory concentration of 6-32 µg/mL with the presence of resistant cells inside the inhibition zone of Etest strips or disks) in multidrug-resistant A. baumannii (MDR-AB). To identify risk factors associated with the emergence of imipenem heteroresistance, a retrospective case-control study was undertaken involving cases with subsequent clinical isolates of the same genotype showing loss of imipenem susceptibility and matched controls with isolates belonging to imipenem-susceptible MDR-AB. The molecular mechanism of heteroresistance was examined. From April 2006 to March 2007, 126 consecutive isolates of MDR-AB were identified from 29 patients. Switch from imipenem susceptibility to heteroresistance was more likely to occur in successive MDR-AB derived from patients who had been exposed to imipenem (length of use 10.9 ± 6.5 days for cases vs. 5.3 ± 4.8 days for controls; P=0.02). An insertion sequence (ISAba1) was found in the promoter region of a class C ß-lactamase gene (bla(ADC-29)) in most imipenem-heteroresistant MDR-AB isolates. In vitro experiments indicated that imipenem heteroresistance, which was associated with overexpression of bla(ADC-29), could be induced by imipenem. Carbapenem use was the only risk factor identified for the emergence of carbapenem-heteroresistant MDR-AB. Physicians should weigh the benefits and risks of each carbapenem-based treatment in managing carbapenem-susceptible MDR-AB infection.

Fecal RNA detection of cytokeratin 19 and ribosomal protein L19 for colorectal cancer.

BACKGROUND/AIMS: We attempted to specifically quantify transcripts of faecal cytokeratin 19 (CK19) and ribosomal protein L19 (RPL19) RNA expression of colorectal cancer and clarify their correlation with clinicopathological parameters and survival in combination. METHODOLOGY: Solid fecal samples were collected and preserved before any treatment. Levels of faecal CK19 and RPL19 mRNA were measured using quantitative real-time PCR. An expression level higher than median value was defined as positive. RESULTS: Between April 2001 and June 2007, 92 patients were recruited. The levels of both markers increased in a trend as stage. Young patients (< 67 years) were correlated with higher rate of CK19+ (p = 0.001), so were higher stages but with borderline significance (p = 0.051). CK19+ and RPL19+ were highly correlated mutually (p = 0.001). Neither CK19+ (p = 0.12) nor RPL19+ (p = 0.14) alone was a prognostic factor of disease-free interval. However, CK19+/RPL19+ was shown to be with worse prognosis (p = 0.037), but not an independent factor in multivariate analysis with stage. CONCLUSIONS: Both markers were significantly higher in the patients of metastatic disease. The use of two markers will recognize the high risk group better than the single marker usage, though not reaching independent status yet. Multi-target strategy assay is suggested for fecal RNA examination.

Clinical meaning of age-related expression of fecal cytokeratin 19 in colorectal malignancy.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of malignant death worldwide. Because young age of onset is often considered a poor prognostic factor for CRC, it is important to identify the poor outcomes of CRC in a younger population and to consider an aggressive approach by implementing early treatment. Our aim was to specifically quantify the fecal cytokeratin 19 (CK19) transcript from CRC patients and investigate its correlation with clinical stage, tumor malignancy, and age. METHODS: The quantitation of fecal CK19 transcript was determined by a quantitative real-time reverse transcription polymerase chain in 129 CRC patients (45 younger than 60 years at diagnosis) and 85 healthy controls. The levels of CK19 protein were examined both in colonic cell lines and tissues. RESULTS: The analysis of 45 younger CRC patients (age

Aggregation of bovine cloned embryos at the four-cell stage stimulated gene expression and in vitro embryo development.

Pre-implantation embryos produced by somatic cell nuclear transfer (SCNT) have varied developmental potentials. The majority of SCNT blastocysts do not develop to term, and the mechanisms inhibiting development are still largely unknown. Aggregation of cloned embryos has been attempted to compensate for the developmental deficiency of individual cloned embryos. In this report, we investigated the impact of aggregation of bovine cloned embryos at the four-cell stage on in vitro development and gene expression of the embryos. Cell numbers and development rate of aggregated (NTagg) and non-aggregated (NT) blastocysts were characterized and compared. The blastocyst formation after aggregation was modeled using the binominal distribution. The results indicate that aggregation enhances the blastocyst formation but does not increase the overall blastocyst rate. Additionally, utilizing microarray gene chip analysis 8.8% of 8,059 genes analyzed were differentially expressed between NTagg and NT blastocysts, with more than 80% of the differentially expressed genes up-regulated in the NTagg blastocysts. Up-regulated genes include those involved in transcription, biosynthesis and signaling such as TDGF1, HNFA, CAV1, GLU5, and CD81. Our results indicate that aggregation of bovine cloned embryos at an early stage promotes the in vitro development of the resulting pre-implantation embryos.

Correlation of K-ras codon 12 mutations in human feces and ages of patients with colorectal cancer (CRC).

Colorectal cancer (CRC) is the predominant gastrointestinal malignancy and constitutes a major medical and economic burden worldwide. A thorough understanding of the oncogenes or genes related to tumorigenesis is the key to developing successful therapeutic strategies. Molecular analysis of feces constitutes a potentially potent and noninvasive method for detection of CRC. Using nested reverse transcription-polymerase chain reaction (RT-PCR) and amplified restriction fragment length polymorphism analysis, sloughed cells from the entire length of the colon and rectum were analyzed for expression of activating K-ras codon 12 mutants, which are becoming attractive targets for antisense treatment. K-ras codon 12 mutant sequences were detected in feces of 5% (1/20) of healthy controls, in feces of 41% (12/29) of CRC patients, in 10% (3/29) of isolates of tissue complementary DNA (cDNA), and in 14% (4/29) of isolates of genomic DNA. Age of patient was significantly associated with K-ras codon 12 sequences in feces: Patients with wild-type K-ras codon 12 sequences were significantly younger than those with mutated forms of K-ras codon 12. Fecal ribonucleic acid (RNA) analysis was demonstrated to be a useful for diagnosis of CRC. This technique may be suitable for screening and determining the clinical significance of active mutations of the K-ras gene in feces and would possibly be useful for identifying patients that would benefit from antisense therapy.

A homologue of the Drosophila headcase protein is a novel tumor marker for early-stage colorectal cancer.

The altered expression of certain genes is frequently detected as a hallmark of malignant tumors. These differentially expressed genes have the potential to become molecular markers. We purified the fecal mRNA from patients with colorectal cancer to identify novel candidates by using oligo-nucleotide microarray hybridization. We identified 21 upregulated and 22 downregulated candidates with significantly altered expression in patient fecal samples that have not been previously characterized. A gene encoding a homologue of the Drosophila headcase protein (HECA) was further examined due to its high ranking and possible importance in some human cancers. A tendency towards increased expression of HECA was dependent upon the clinicopathological stage. In this report, healthy individuals expressed less HECA, either in their blood samples or feces. Moreover, we detected upregulated HECA in blood and fecal samples of patients with colorectal cancer, and its expression level was shown to be significantly correlated with disease status. Our data show that HECA may be an early-stage classifier of colorectal cancer that can discriminate between late- and early-stage disease. In conclusion, this study is the first to analyze differentially expressed genes in the feces of colorectal cancer patients using oligonucleotide microarrays. The data suggest that HECA expression levels in feces may be an effective classifier for early-stage colorectal cancer.

Management of bile leakage after laparoscopic cholecystectomy based on etiological classification.

PURPOSE: To analyze and classify bile leakage after laparoscopic cholecystectomy (LC) according to its etiology. This classification will help to determine the most appropriate management strategy, whereby unnecessary intervention can be avoided. METHODS: We examined the medical records of 16 patients in whom bile leakage occurred as a complication of LC. RESULTS: Bile leakage was classified according to its cause into the following groups: insecure closure of the cystic duct stump (n = 3); retention of a common bile duct (CBD) stone (n = 1); CBD injury (n = 10); unsuspected accessory bile ducts (n = 1); and unknown origin (n = 1). The management strategies included observation (n = 3), laparoscopic intervention with drainage (n = 4), laparotomy with drainage (n = 3), and laparotomy with Roux-en-Y choledochojejunostomy for CBD transection (n = 6). All 16 patients recovered uneventfully with similar hospitalization. CONCLUSIONS: Bile leakage is not always caused by bile duct injury, and it would be inappropriate to attribute leakage to bile duct injury if there is a retained CBD stone, an unsuspected accessory duct, or an unsecured cystic duct stump. Thus, the management of each condition should vary accordingly. Reviewing a videotape of the surgery and early cholangiogram can help to establish the etiological diagnosis and select the most appropriate course of action.

Production of alpha 1,3-galactosyltransferase-deficient pigs.

The enzyme alpha1,3-galactosyltransferase (alpha1,3GT or GGTA1) synthesizes alpha1,3-galactose (alpha1,3Gal) epitopes (Galalpha1,3Galbeta1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of alpha1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the alpha1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the alpha1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the alpha1,3GT protein. Four healthy alpha1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the alpha1,3GT gene hold significant value, as they would allow production of alpha1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.

Efficient production of transgenic cloned calves using preimplantation screening.

The genetic manipulation of donor cells before nuclear transfer (NT) enables prior selection for transgene integration. However, selection for genetically modified cells using antibiotic drugs often results in mixed populations, resulting in a mixture of transgenic and nontransgenic donor cells for NT. In this study, we attempted to develop efficient strategies for the generation of human bile salt-stimulated lipase (BSSL) transgenic cows. Preimplantation screening by either biopsy or green fluorescent protein (GFP) expression was used to detect NT-derived BSSL transgenic embryos to ensure that the calf born would be transgenic. We compared the development rates of NT-derived embryos from G418- and GFP-selected donor cells. There were no significant differences (P < 0.001) in cleavage rate (67.2% vs. 60.0%) and blastocyst formation rate (44.9% vs. 41.2%). We also compared the pregnancy rates of the G418/biopsy and GFP preimplantation screened NT-derived blastocysts. The Day 40 pregnancy rate of the G418/biopsy group (40%) was lower than that of the GFP group (57%), but the calf birth rate of the G418/biopsy group (40%) was higher than that of the GFP group (21%). Healthy BSSL transgenic calves were born after both screening processes. This is the first report of biopsy-screened cloned transgenic animals. The results suggest that both selection methods are useful for detecting transgenic NT embryos without negatively affecting their development into viable transgenic offspring.