Exploring associations between the teat apex metagenome and Staphylococcus aureus intramammary infections in primiparous cows under organic directives.

The primary objective of this study was to identify associations between the prepartum teat apex microbiome and the presence of Staphylococcus aureus intramammary infections (IMI) in primiparous cows during the first 5 weeks after calving. We performed a case-control study using shotgun metagenomics of the teat apex and culture-based milk data collected longitudinally from 710 primiparous cows on five organic dairy farms. Cases had higher odds of having S. aureus metagenomic DNA on the teat apex prior to parturition compared to controls (OR = 38.9, 95% CI: 14.84-102.21). Differential abundance analysis confirmed this association, with cases having a 23.8 higher log fold change (LFC) in the abundance of S. aureus in their samples compared to controls. Of the most prevalent microorganisms in controls, those associated with a lower risk of post-calving S. aureus IMI included Microbacterium phage Min 1 (OR = 0.37, 95% CI: 0.25-0.53), Corynebacterium efficiens (OR = 0.53, 95% CI: 0.30-0.94), Kocuria polaris (OR = 0.54, 95% CI: 0.35-0.82), Micrococcus terreus (OR = 0.64, 95% CI: 0.44-0.93), and Dietzia alimentaria (OR = 0.45, 95% CI: 0.26-0.75). Genes encoding for Microcin B17 AMPs were the most prevalent on the teat apex of cases and controls (99.7% in both groups). The predicted abundance of genes encoding for Microcin B17 was also higher in cases compared to controls (LFC 0.26). IMPORTANCE: Intramammary infections (IMI) caused by Staphylococcus aureus remain an important problem for the dairy industry. The microbiome on the external skin of the teat apex may play a role in mitigating S. aureus IMI risk, in particular the production of antimicrobial peptides (AMPs) by commensal microbes. However, current studies of the teat apex microbiome utilize a 16S approach, which precludes the detection of genomic features such as genes that encode for AMPs. Therefore, further research using a shotgun metagenomic approach is needed to understand what role prepartum teat apex microbiome dynamics play in IMI risk.

Predictive models for metritis cure using farm-collected data, metabolic and inflammation biomarkers, and hemogram variables measured at diagnosis.

Our objective was to evaluate the accuracy of predictive models for metritis spontaneous cure (SC) and cure among ceftiofur-treated cows using farm-collected data only, and with the addition of hemogram variables and circulating concentration of metabolites, minerals, and biomarkers (BM) of inflammation measured at time of diagnosis. Data related to parity, calving-related issues, BCS, rectal temperature, and DIM at metritis diagnosis were collected from a randomized clinical trial that included 422 metritic cows from 4 herds in Texas, California, and Florida. Metritis was defined as the presence of red-brownish, watery, and fetid vaginal discharge, and cure was defined as the absence of metritis 14 d after initial diagnosis. Cows were randomly allocated to receive systemic ceftiofur therapy (2 subcutaneous doses of 6.6 mg/kg of ceftiofur crystalline-free acid on the day of diagnosis and 3 d later; CEF) or to remain untreated (control). At enrollment (day of metritis diagnosis), blood samples were collected and submitted to complete blood count (CBC) and processed for the measurement of 13 minerals and BM of metabolism and inflammation. Univariable analysis to evaluate the association of farm-collected data and blood-assessed variables with metritis cure were performed, and variables with P ≤ 0.20 were offered to multivariable logistic regression models and retained if P ≤ 0.15. The areas under the curve for models predicting SC using farm data only and farm + BM were 0.70 and 0.76, respectively. Complete blood count variables were not retained in the models for SC. For models predicting cure among CEF cows, the area under the curve was 0.75, 0.77, 0.80, and 0.80 for models using farm data only, farm + CBC, farm + BM, and farm + CBC + BM, respectively. Predictive models of metritis cure had fair accuracy, with SC models being less accurate than models predictive of cure among CEF cows. Additionally, adding BM variables marginally improved the accuracy of models using farm collected data, and CBC data did not improve the accuracy of predictive models.

A randomized controlled trial evaluating the efficacy of systemic ceftiofur administration for metritis therapy in dairy cows and the effect of metritis cure on economically important outcomes.

The main objective of this study was to evaluate the effect of ceftiofur on metritis cure, milk yield, reproductive performance, and culling up to 300 DIM. The secondary objective was to evaluate the effect of metritis cure at 5 (ECURE) and 14 (LCURE) d after diagnosis on milk production, reproduction, and culling. A total of 422 Holstein cows diagnosed with metritis from 4 herds located in TX, CA, and FL were enrolled in a randomized clinical trial. Cows diagnosed with metritis (fetid, watery, reddish/brownish uterine discharge) were blocked by herd and parity and were randomly allocated to receive systemic administration of ceftiofur (CEF) or to remain untreated (CON). In addition, 399 non-metritic cows (NMET) were included for comparison purposes. Metritis cure was evaluated at 5 and 14 d after diagnosis and was defined as the absence of metritis clinical signs. Logistic regression models were fitted to the data to assess the effect of treatment on metritis cure. Milk yield was analyzed using a mixed linear model, while logistic regression, Cox proportional hazard and Kaplan-Meier survival analysis models were fitted to culling and reproduction data. Cows treated with CEF had 1.86 (95% CI: 1.22 - 2.81) and 1.68 (95% CI: 1.02 - 2.75) greater odds of being cured than CON cows at 5 and 14 d after diagnosis, respectively. No effect of CEF was observed for milk yield; however, NMET cows had greater milk yield compared with metritic cows (CEF = 36.0, 95% CI = 33.8 - 38.1; CON = 36.1, 95% CI = 33.9 - 38.2; NMET = 36.9 kg/d, 95% CI = 34.8 - 39.4). Likewise, no effect of CEF was observed on reproductive performance and culling. Nonetheless, the likelihood of conceiving for NMET cows was 1.72 (95% CI = 1.41 - 2.12) and 1.64 (95% CI = 1.33 - 2.00) times greater than for CEF and CON cows, respectively. Ceftiofur-treated and CON cows had 2.93 (95% CI = 1.90 - 4.51) and 2.37 (95% CI = 1.51 - 3.71) greater hazard of culling compared with NMET, respectively. Regardless of treatment, no differences between ECURE and LCURE were observed on milk yield, reproduction, and culling throughout the entire lactation, but cows that cured at 5 or 14 d after diagnosis had greater milk production in the first 60 DIM compared with cows that did not cure (NCURE). Cows in ECURE and LCURE also had a 1.59 (95% CI = 1.16 - 2.16) and 1.49 (95% CI = 1.08 - 2.05) greater hazard of pregnancy and 0.43 (95% CI = 0.26-0.71) and 0.56 (95% CI = 0.34-0.92) hazard of culling compared with NCURE. Ceftiofur therapy increased metritis cure, but benefits to productivity and longevity were not observed. Also, cows that fail to cure have impaired lactation performance, but no differences regarding timing of cure were observed.

Effect of subclinical mastitis detected in the first month of lactation on somatic cell count linear scores, milk yield, fertility, and culling of dairy cows in certified organic herds.

It is well established that subclinical mastitis (SCM), characterized by somatic cell count (SCC) >200,000 cells/mL, has a negative effect on the productivity, reproductive performance, and survivability of cows from conventional dairy herds. However, in organic herds, where the use of antimicrobial drugs is restricted for the treatment and control of intramammary infections (IMI) in dairy cows, little is known about the effect of SCM on performance and survivability. The objective of this study was to evaluate whether SCM diagnosed during the first month of lactation was associated with SCC linear score dynamics, milk production, fertility, and culling of dairy cows in USDA-certified organic herds. We collected data from 2 organic herds in New Mexico and Texas. A total of 1,511 cows that calved between June 2018 and May 2019 were included in the study and were followed until month 10 of the current lactation. Cows with SCC >200,000 cells/mL in the first month of lactation were considered to have SCM. We used mixed linear regression models accounting for repeated measures to assess the effect of SCM on monthly milk production and SCC linear scores. We used Cox proportional hazards models to evaluate the effect of SCM on the risk of pregnancy and culling. We considered parity, farm, previous gestation length, stillbirth, twinning, dystocia, and 2- and 3-way interactions as potential confounders. Cows diagnosed with SCM during the first month of lactation produced less milk than cows without SCM. Cows with SCM had elevated SCC linear scores during their previous lactation and throughout the subsequent months of lactation compared to cows without SCM. The effect of SCM on SCC linear scores was more pronounced in multiparous than primiparous cows. Subclinical mastitis during the first month of lactation did not affect the likelihood of pregnancy during the first 300 d in milk. Cows with SCM in the first month were more likely to die or be culled during the 300 d of lactation than cows without SCM. We observed that elevated SCC in the first month of lactation had detrimental effects on the milk yield and survivability of dairy cows in USDA organic herds, but it did not affect reproductive performance. We demonstrated that cows with SCM diagnosed in the first month of lactation continued to have elevated SCC linear scores throughout their entire lactation, and that elevated SCC was carried over from the previous lactation.

Investigating the cow skin and teat canal microbiomes of the bovine udder using different sampling and sequencing approaches.

There is a need for standardized, efficient, and practical sampling methods to support large population-based studies of the internal and external epithelial microbiomes of the bovine udder. The primary objective of this study was to evaluate different sampling devices for the isolation of microbial DNA originating from the internal and external teat epithelium. Secondary objectives were to survey and compare the microbial diversity of external and teat canal epithelial microbiomes using amplicon and shotgun metagenomic sequencing approaches. To address these objectives, we enrolled a convenience sample of 24 Holstein dairy cows and collected samples from the external epithelium at the base of udder, the external teat barrel epithelium, the external teat apex epithelium, and the teat canal epithelium. Extracted DNA was quantified and subjected to PCR amplification of the V4 hypervariable region of the 16S rRNA gene and sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, CA). A subset of samples was subjected to a shallow shotgun metagenomic assay on the Illumina HiSeq platform. For samples collected from the external teat epithelium, we found that gauze squares consistently yielded more DNA than swabs, and Simpson's reciprocal index of diversity was higher for gauze than for swabs. The teat canal epithelial samples exhibited significantly lower diversity than the external sampling locations, but there were no significant differences in diversity between teat apex, teat barrel, and base of the udder samples. There were, however, differences in the microbial distribution and abundances of specific bacteria across external epithelial surfaces. The proportion of shotgun sequence reads classified as Bos taurus was highly variable between sampling locations, ranging from 0.33% in teat apex samples to 99.91% in teat canal samples. These results indicate that gauze squares should be considered for studying the microbiome of the external epithelium of the bovine udder, particularly if DNA yield must be maximized. Further, the relative proportion of host to non-host DNA present in samples collected from the internal and external teat epithelium should be considered when designing studies that utilize shotgun metagenomic sequencing.

The 2019 PGDIS position statement on transfer of mosaic embryos within a context of new information on PGT-A.

BACKGROUND: A recently published Position Statement (PS) by the Preimplantation Genetics Diagnosis International Society (PGDIS) regarding utilization of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF) contained inaccuracies and misrepresentations. Because opinions issued by the PGDIS have since 2016 determined worldwide IVF practice, corrections appear of importance. METHODS: The International Do No Harm Group in IVF (IDNHG-IVF) is a spontaneously coalesced body of international investigators, concerned with increasing utilization of add-ons to IVF. It is responsible for the presented consensus statement, which as a final document was reached after review of the pertinent literature and again revised after the recent publication of the STAR trial and related commentaries. RESULTS: In contrast to the PGDIA-PS, we recommend restrictions to the increasing, and by IVF centers now often even mandated, utilization of PGT-A in IVF cycles. While PGT-A has been proposed as a tool for achieving enhanced singleton livebirth outcomes through embryo selection, continued false-positive rates and increasing evidence for embryonic self-correction downstream from the testing stage, has led IDNHG-IVF to conclude that currently available data are insufficient to impose overreaching recommendations for PGT-A utilization. DISCUSSION: Here presented consensus offers an alternative to the 2019 PGDIS position statement regarding utilization of preimplantation genetic testing for aneuploidy (PGT-A) in association with in vitro fertilization (IVF). Mindful of what appears to offer best outcomes for patients, and in full consideration of patient autonomy, here presented opinion is based on best available evidence, with the goal of improving safety and efficacy of IVF and minimizing wastage of embryos with potential for healthy births. CONCLUSIONS: As the PGDIS never suggested restrictions on clinical utilization of PGT-A in IVF, here presented rebuttal represents an act of self-regulation by parts of the IVF community in attempts to control increasing utilization of different unproven recent add-ons to IVF.

Modelling considerations in the analysis of associations between antimicrobial use and resistance in beef feedlot cattle.

A number of sophisticated modelling approaches are available to investigate potential associations between antimicrobial use (AMU) and resistance (AMR) in animal health settings. All have their advantages and disadvantages, making it unclear as to which model is most appropriate. We used advanced regression modelling to investigate AMU-AMR associations in faecal non-type-specific Escherichia coli (NTSEC) isolates recovered from 275 pens of feedlot cattle. Ten modelling strategies were employed to investigate AMU associations with resistance to chloramphenicol, ampicillin, sulfisoxazole, tetracycline and streptomycin. Goodness-of-fit statistics did not show a consistent advantage for any one model type. Three AMU-AMR associations were significant in all models. Recent parenteral tetracycline use increased the odds of finding tetracycline-resistant NTSEC [odds ratios (OR) 1·1-3·2]; recent parenteral sulfonamide use increased the odds of finding sulfisoxazole-resistant NTSEC (OR 1·4-2·5); and recent parenteral macrolide use decreased the odds of recovering ampicillin-resistant NTSEC (OR 0·03-0·2). Other results varied markedly depending on the modelling approach, emphasizing the importance of exploring and reporting multiple modelling methods based on a balanced consideration of important factors such as study design, mathematical appropriateness, research question and target audience.

The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

Cow-related factors associated with intramammary infections during the 1st week postpartum in primiparous dairy cows in certified organic herds.

The objectives if this exploratory study were to identify variables associated with intramammary infections (IMI) during the 1st week of lactation in primiparous organic dairy cows, and to evaluate the association of those variables with somatic cell count (SCC) linear scores and milk yield in early lactation. Nulliparous cows (n = 240) were evaluated for: udder edema, teat edema, milk leakage (ML) and udder hygiene at weeks 6, 4, 2, and 1 prepartum and 1st week postpartum; body condition score (BCS) at 6 weeks prepartum and 1st week postpartum; age at calving (days), gestation length, dystocia, stillbirth, calf sex were included in the analysis. Milk samples from the 1st week postpartum were cultured using standard laboratory techniques and bacterial growth was considered IMI. Intramammary infection was observed in 58.7% of cows, with Staphylococcus chromogenes and S. aureus being the most prevalent pathogens. Cows with ML at 1st week postpartum were at 3.42 greater odds of IMI (P < 0.01). Cows with prepartum BCS ≥ 3.75 were at 3.12 greater odds of IMI (P < 0.01). Cows with ML in the 1st week of lactation had increased SCC (P < 0.01) and lower milk production in the second month of lactation (P = 0.05). Intervention studies are needed to evaluate if monitoring prepartum BCS and ML at early postpartum can aid in the control of IMIs in heifers in organic dairies.

Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies.

Over the past decade, the number of reported live births resulting from oocyte cryopreservation has rapidly increased. To appreciate the true number of children born, verified live births were tabulated and assessed. A literature search was performed; authors were then contacted to verify birth outcomes and provide updates. A database including all verified live born infants was constructed. A total of 58 reports (1986-2008) were reviewed, which included 609 live born babies (308 from slow freezing, 289 from vitrification and 12 from both methods). Additionally, 327 other live births were verified. Of the total 936 live borns, 1.3% (12) were noted to have birth anomalies: three ventricular septal defects, one choanal and one biliary atresia, one Rubinstein-Taybi syndrome, one Arnold-Chiari syndrome, one cleft palate, three clubfoot and one skin haemangioma. Compared with congenital anomalies occurring in naturally conceived infants, no difference was noted. With more live born data accumulating, this procedure may become mainstream as a fertility preservation option, particularly for women diagnosed with malignancy requiring cytotoxic therapy. A registry would help to assure the safest, most expeditious development of this technology.

Mannheimia haemolytica in feedlot cattle: prevalence of recovery and associations with antimicrobial use, resistance, and health outcomes.

BACKGROUND: Mannheimia haemolytica is an important etiological agent in bovine respiratory disease. OBJECTIVES: Explore risk factors for recovery of susceptible and resistant M. haemolytica in feedlot cattle and explore associations with health outcomes. ANIMALS: Cattle (n = 5,498) from 4 feedlots sampled at arrival and later in feeding period. METHODS: Susceptibility of M. haemolytica isolates tested for 21 antimicrobials. Records of antimicrobial use and health events analyzed using multivariable regression. RESULTS: M. haemolytica recovered from 29% of cattle (1,596/5,498), 13.1% at arrival (95% CI, 12.3-14.1%), and 19.8% at second sampling (95% CI, 18.7-20.9%). Nearly half of study cattle received antimicrobial drugs (AMDs) parenterally, mostly as metaphylactic treatment at arrival. Individual parenteral AMD exposures were associated with decreased recovery of M. haemolytica (OR, 0.2; 95% CI, 0.02-1.2), whereas exposure in penmates was associated with increased recovery (OR, 1.5; 95% CI, 1.05-2.2). Most isolates were pan-susceptible (87.8%; 95% CI, 87.0-89.4%). AMD exposures were not associated with resistance to any single drug. Multiply-resistant isolates were rare (5.9%; 95% CI, 5.1-6.9%), but AMD exposures in pen mates were associated with increased odds of recovering multiply-resistant M. haemolytica (OR, 23.9; 95% CI, 8.4-68.3). Cattle positive for M. haemolytica on arrival were more likely to become ill within 10 days (OR, 1.7; 95% CI, 1.1-2.4). CONCLUSIONS AND CLINICAL IMPORTANCE: Resistance generally was rare in M. haemolytica. Antimicrobial drug exposures in penmates increased the risk of isolating susceptible and multiply-resistant M. haemolytica, a finding that could be explained by contagious spread.

Rapid Salmonella detection in experimentally inoculated equine faecal and veterinary hospital environmental samples using commercially available lateral flow immunoassays.

REASONS FOR PERFORMING STUDY: Salmonella enterica is the most commonly reported cause of outbreaks of nosocomial infections in large animal veterinary teaching hospitals and the closure of equine hospitals. Rapid detection may facilitate effective control practices in equine populations. Shipping and laboratory testing typically require ≥48 h to obtain results. Lateral flow immunoassays developed for use in food-safety microbiology provide an alternative that has not been evaluated for use with faeces or environmental samples. OBJECTIVES: We aimed to identify enrichment methods that would allow commercially available rapid Salmonella detection systems (lateral flow immunoassays) to be used in clinical practice with equine faecal and environmental samples, providing test results in 18-24 h. STUDY DESIGN: In vitro experiment. METHODS: Equine faecal and environmental samples were inoculated with known quantities of S. enterica serotype Typhimurium and cultured using 2 different enrichment techniques for faeces and 4 enrichment techniques for environmental samples. Samples were tested blindly using 2 different lateral flow immunoassays and plated on agar media for confirmatory testing. RESULTS: In general, commercial lateral flow immunoassays resulted in fewer false-negative test results with enrichment of 1 g faecal samples in tetrathionate for 18 h, while all environmental sample enrichment techniques resulted in similar detection rates. The limit of detection from spiked samples, ∼4 colony-forming units/g, was similar for all methods evaluated. CONCLUSIONS: The lateral flow immunoassays evaluated could reliably detect S. enterica within 18 h, indicating that they may be useful for rapid point-of-care testing in equine practice applications. Additional evaluation is needed using samples from naturally infected cases and the environment to gain an accurate estimate of test sensitivity and specificity and to substantiate further the true value of these tests in clinical practice.

Hysteroscopic cervical canal shaving: a new therapy for cervical stenosis before embryo transfer in patients undergoing in vitro fertilization.

OBJECTIVE: To report a case of cervical stenosis repaired by hysteroscopic cervical shaving that created a smooth passage for ET in a patient undergoing IVF. DESIGN: Case report. SETTING: University-based IVF program. PATIENT(S): A woman with a history of multiple failed IVF attempts in whom ET was extremely difficult. INTERVENTION(S): Operative hysteroscopy with creation of a cervical tract. MAIN OUTCOME MEASURE(S): Ease of postoperative ET and outcome of IVF treatment after the hysteroscopic procedure. RESULT(S): Easy performance of ET and a resulting triplet pregnancy. CONCLUSION(S): This novel hysteroscopic repair of cervical stenosis resulted in a markedly easier ET and a viable pregnancy.

Massive deep vein thrombosis in a patient with antithrombin III deficiency undergoing ovarian stimulation for in vitro fertilization.

OBJECTIVE: To present the first report of a thromboembolic complication in early pregnancy after ovarian hyperstimulation for IVF in a patient with AT III deficiency who was treated successfully and subsequently delivered a healthy male infant at 32 weeks of gestation. DESIGN: Case report. SETTING: Hospital-based clinic for reproductive medicine. PATIENT: A 28-year-old woman who consulted our IVF clinic with a 3.5-year history of primary infertility. INTERVENTIONS: Intravenous heparin therapy. RESULTS: The patient responded adequately to heparin therapy and was discharged home on SC heparin. A primary cesarean section was performed at 32 weeks of gestation because of poor fetal growth and transverse lie. CONCLUSIONS: We stress the importance of obtaining a thorough personal and family history before initiation of ovarian hyperstimulation. Measuring activity of AT III, protein C, and protein S in patients with a suspicious history of thromboembolic episodes occurring at an early age may lead to the implementation of appropriate prophylactic measures, preventing potentially life-threatening complications.

Assisted hatching facilitates earlier implantation.

OBJECTIVE: To examine whether opening of the zona pellucida (i.e., assisted hatching) accelerates implantation. DESIGN: In a controlled, randomized trial, patients were assigned to control and assisted hatching groups. SETTING: All patients studied were of the Center for Reproductive Medicine at Cornell University Medical College. INTERVENTION: All patients underwent stimulation with gonadotropins after luteal phase GnRH down regulation. Assisted hatching with zona drilling using acidic Tyrode's solution was performed on the assigned embryos. MAIN OUTCOME MEASURES: Luteal E2, P, and hCG on days +5, +6, +7, +8, +9, +11, +13, and +15 were measured. The implantation time, peak midluteal E2 and intervals between these two values were studied. RESULTS: Implantation occurred significantly earlier in the assisted hatching group. The interval between implantation and peak midluteal E2 was also significantly shorter in the assisted hatching group than in the controls. However, there was no significant difference in the day of the peak midluteal E2 between the assisted hatching and control groups. CONCLUSION: Assisted hatching may enhance embryo implantation not only by mechanically facilitating the hatching process but also by allowing earlier embryo-endometrium contact. Such early contact may enhance embryonic development potential and may optimize synchronization between embryo and endometrium, resulting in improved implantation efficiency.

In vitro fertilization outcome relative to embryo transfer difficulty: a novel approach to the forbidding cervix.

OBJECTIVE: To assess the impact of ET difficulty on IVF outcome and to optimize the ET procedure. DESIGN: Retrospective analysis of IVF outcome by ET catheter type and ET difficulty. Prospective treatment and follow-up of patients with a history of extremely difficult cervical passage. SETTING: Large university-based IVF program. PATIENT(S): All patients < 40 years of age undergoing IVF-ET from September 1995 to May 1998. INTERVENTION(S): Surgical correction of cervical stenosis. MAIN OUTCOME MEASURE(S): Pregnancy and embryo implantation rates. RESULT(S): Only 0.6% of ETs were "extremely difficult." Pregnancy rates were not statistically significantly different among ETs graded easy, moderate, and difficult. In contrast, no pregnancies occurred in the rare "extremely difficult" ET group. Eight patients with a history of extremely difficult cervical passage underwent surgical correction of their cervical stenosis. Twelve postoperative IVF-ET in these women resulted in eight clinical pregnancies, six of which were multiple gestations. The embryo implantation rate of these cycles was 42.2%. CONCLUSION(S): Patients with a history of extremely difficult ET may benefit from hysteroscopic evaluation and possible modification of their cervical canal before a future IVF attempt.

A two- versus three-embryo transfer: the oocyte donation model.

OBJECTIVE: To compare implantation and pregnancy rates in oocyte recipients undergoing a two-embryo versus three-embryo transfer, 3 days after retrieval. DESIGN: Retrospective comparative analysis. SETTING: University-based in vitro fertilization center. PATIENT(S): All oocyte recipients undergoing embryo transfer from January 1, 1997 through August 31, 1999. INTERVENTION(S): Recipients received two or three embryos. MAIN OUTCOME MEASURE(S): Implantation, and clinical and multiple pregnancy rates. RESULT(S): Seventy-three recipients underwent a two-embryo transfer, and 376 had three embryos replaced. The numbers of oocytes retrieved (12.7 +/- 0.89 vs. 13.1 +/- 0.36) and embryos obtained (8.05 +/- 0.65 vs. 8.77 +/- 0.27) did not differ between the two-embryo and three-embryo transfer groups, nor did the proportion of patients with embryo cryopreservation (54.3% vs. 42.6%, respectively). There was no significant difference in pregnancy or implantation rates when comparing those patients with a two-embryo transfer to those with a three-embryo transfer. Significantly, 13.8% of the pregnancies in the three-embryo transfer group were triplet. CONCLUSION(S): Reducing the number of embryos transferred in an oocyte donation cycle can lower the incidence of triplet pregnancies without significantly lowering the overall pregnancy rate.

Elevated day 3 serum follicle stimulating hormone and/or estradiol may predict fetal aneuploidy.

OBJECTIVE: To determine whether baseline serum FSH and/or E2 concentrations can predict the risk for fetal chromosomal abnormalities. DESIGN: Case control study. SETTING: Reproductive technology program at a university hospital. PATIENT(S): Patients who underwent dilation and curettage (D + C), and whose products of conception were karyotyped. INTERVENTION(S): Patients underwent natural conception or controlled ovarian hyperstimulation followed by intrauterine insemination, in vitro fertilization and embryo transfer, gamete intrafallopian transfer, or zygote intrafallopian transfer. MAIN OUTCOME MEASURE(S): Baseline serum FSH and E2 concentrations and fetal karyotype. RESULT(S): Genetic evaluation of 78 D + C specimens revealed 34 normal and 44 abnormal fetal karyotypes. A significantly greater proportion of women with abnormal fetal karyotype had elevated baseline serum FSH (> or =15 mIU/mL [RIA] or 10 mIU/mL [Immulite]) and/or E2 > or = 50 pg/mL [Immulite]) compared with women of normal fetal karyotype. Among karyotypically abnormal abortuses, autosomal trisomy was the most common abnormality noted (79.5%), followed by mosaicism (6.8%), triploidy (6.8%), monosomy XO (4.5%), and balanced translocation (2.3%). CONCLUSION(S): Baseline serum FSH and/or E2 concentrations may be valuable as predictors of fetal aneuploidy.

Enhancement or initiation of testicular sperm motility by in vitro culture of testicular tissue.

OBJECTIVE: To describe different techniques of testicular tissue culture and their effect on sperm motility, mainly in cases of totally immotile spermatozoa, and to compare the effect of in vitro culture with that of motility stimulants. DESIGN: Prospective study. SETTING: University teaching hospital. PATIENT(S): Ten patients undergoing testicular biopsy for diagnostic purposes or for intracytoplasmic sperm injection. INTERVENTION(S): Dissected testicular biopsy samples and tissue blocks were cultured at 37 degrees C for up to 96 hours. Immediately after dissection, immotile testicular spermatozoa were incubated for 30 minutes in pentoxifylline and 2-deoxyadenosine. MAIN OUTCOME MEASURE(S): Sperm motility and vitality. RESULT(S): Overall, dissected samples showed improved sperm motility, which peaked within 48 hours of culture. Unlike motility, vitality declined linearly, from 56.3%+/-19% at initial assessment to 18.8%+/-11% at 96 hours. Five samples had initially immotile spermatozoa, of which four acquired motility at 48 hours. In vitro culture showed results comparable with those of incubation with pentoxifylline and 2-deoxyadenosine. Culture of tissue blocks did not improve motility or vitality compared with dissected tissue. CONCLUSION(S): The motility of testicular spermatozoa was enhanced or initiated after in vitro culture. Testicular biopsy culture may be an alternative to the use of motility stimulants to obtain motile spermatozoa for intracytoplasmic sperm injection, particularly when oocytes are not immediately available.