Trophectoderm biopsy protocols can affect clinical outcomes: time to focus on the blastocyst biopsy technique.

OBJECTIVE: To compare two different blastocyst biopsy protocols. DESIGN: Retrospective single-center cohort study. SETTINGS: Private in vitro fertilization center. PATIENT(S): The study included 1,670 frozen-thawed embryo transfers (FETs) with preimplantation genetic testing for aneuploidy (PGT-A). INTERVENTION: None. MAIN OUTCOME MEASURE(S): Survival rate (SR) after thawing, clinical pregnancy rate (CPR), ongoing implantation rate (IR), and live birth rate (LBR). RESULT(S): Eight hundred thirty-five FETs with PGT-A cycles including only embryos biopsied in the sequential blastocyst hatching and biopsy protocol paired with the ablation of one-fourth of the zona pellucida (ZP) were matched with 835 FETs with PGT-A cycles including only embryos biopsied in the day 3 prehatching protocol by female age (±1 year), number of embryos transferred, use of gestational carrier or egg donor, and day of blastocyst transfer. Only FETs with euploid blastocysts graded no lower than 4BB were included, and cycles with fewer than five oocytes were excluded. SR after thawing, CPR, ongoing IR, and LBR were significantly higher in the FET cycles with the embryos biopsied in the sequential hatching and biopsy protocol. Four cases of monozygotic twin pregnancies were reported with the day 3 prehatching protocol and none with the sequential hatching and biopsy protocol. CONCLUSION(S): Our results show, for the first time, that using different blastocyst biopsy protocols can affect clinical outcomes. Because the study was retrospective, our findings should be validated in a prospective trial.

Controlled ovarian hyperstimulation (COH) parameters associated with euploidy rates in donor oocytes.

Although oocyte donors are young and are expected to provide a high rate of euploid oocytes, significant differences of euploidy rates for donor embryos exist between different IVF centers (1). Laboratory conditions can lead to differences of euploidy (2,3,4,5,6,7); but, the role of COH has not been investigated. In this study, we investigated whether euploidy rates in the embryos created from donor oocytes are influenced by controlled ovarian hyperstimulation parameters used during assisted reproduction. Euploidy rates in egg donor cycles undergoing PGT-A (N = 423) were examined retrospectively for associations with donor age, gonadotropin doses (dose per day), the fraction of gonadotropin provided by hMG (F(hMG)), days of stimulation, estradiol per mature oocyte on day of trigger, number of mature oocytes retrieved, number of embryos biopsied, incidence of euploidy and physician of record. Differences in euploidy rates between physicians were examined using analysis of variance. The proportion of euploid embryos per donor cycle was examined for associations with COH parameters using pairwise post-hoc comparisons, adjusting for multiple testing. The set of variables from this analysis was then submitted to a principal component analysis. Linear regression analysis was used to assess the relationships between stimulation parameters and the incidence of euploidy (the dependent variable). Euploidy rates and cycle parameters varied significantly among treating physicians. Euploidy rates (expressed as a fraction of biopsied embryos) were associated (p = 0.01) only with the F(hMG) but not with the number of MII retrieved or other variables. On the other hand, the number of euploid embryos (in contrast to the euploidy rate) was associated with the number of MII produced. Donor euploidy rates are significantly associated with the fraction of total gonadotropin comprising human menopausal gonadotropin (or F(hMG)) during controlled ovarian hyperstimulation but are not associated with other cycle parameters. The study provides the first suggestion that patient stimulation parameters can affect the incidence of euploidy in embryos generated through the use of standard assisted reproductive techniques. The study is limited by its retrospective approach and because the aCGH analysis used is less sensitive than more recent NGS technology. Further, it provides a suggestion that the use of hMG is beneficial for obtaining euploid embryos.

Impact of a prior cesarean delivery on embryo transfer: a prospective study.

OBJECTIVE: To determine whether a history of prior cesarean delivery (CD) makes ET more difficult and impacts pregnancy outcomes. DESIGN: Prospective cohort study. SETTING: Tertiary care military facility. PATIENT(S): One hundred ninety-four patients with previous delivery undergoing IVF/intracytoplasmic sperm injection (ICSI)-ET. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Live birth (primary), positive hCG, clinical pregnancy, and time to perform ET. RESULT(S): There was no statistically significant difference between patients with a history of only vaginal deliveries versus those with a history of CD for live birth (39% vs. 32%), positive hCG (56% vs. 53%), or clinical pregnancy (49% vs. 41%). Embryo transfers took longer in the history of CD group (157 vs. 187 seconds) and were more likely to have mucus (27% vs. 45%) or blood (8% vs. 21%) on the catheter. CONCLUSION(S): Embryo transfers performed on patients with a prior CD took 30 seconds longer. They were also more likely to have blood or mucus on the catheter. Despite the apparently more difficult transfers, pregnancy outcomes were not different between the two groups.

Utility of antimüllerian hormone level obtained for infertility assessment for alerting to ovarian tumor.

BACKGROUND: Antimüllerian hormone is a marker of ovarian reserve and is now routinely measured in women seeking infertility treatment. CASE: A 42-year-old woman, gravida 1, abortus 1, presented with secondary infertility. Obtained originally for assessment of ovarian reserve, an antimüllerian hormone of more than 160 ng/mL raised suspicion for a granulosa cell tumor. A laparoscopic right salpingo-oophorectomy, pelvic washings, dilation and curettage, and peritoneal and omental biopsies were performed. A well-differentiated granulosa cell tumor confined to the right ovary was consistent with stage 1A disease. CONCLUSION: As the use of antimüllerian hormone becomes more common for ovarian reserve testing, providers need to maintain an awareness for neoplastic disease with abnormal values.

Falsely elevated testosterone due to heterophile antibodies.

BACKGROUND: Falsely elevated testosterone is a rare phenomenon that may result from heterophile antibodies. Similar to patients with heterophile antibodies for ß-hCG, incorrect management may result in unnecessary testing or therapy. CASE: A previously healthy postmenopausal woman presented with a falsely elevated total testosterone level due to interference consistent with heterophile antibodies, with subsequent normal levels detected by liquid chromatography-mass spectrometry. CONCLUSION: Asymptomatic patients may present with an elevated hormone level due to heterophile antibody interference. Molecular studies for hormone levels can assist greatly when falsely elevated levels are suspected, but these tests are costly and time-intensive.

Characterization of tissue biomechanics and mechanical signaling in uterine leiomyoma.

Leiomyoma are common tumors arising within the uterus that feature excessive deposition of a stiff, disordered extracellular matrix (ECM). Mechanical stress is a critical determinant of excessive ECM deposition and increased mechanical stress has been shown to be involved in tumorigenesis. Here we tested the viscoelastic properties of leiomyoma and characterized dynamic and static mechanical signaling in leiomyoma cells using three approaches, including measurement of active RhoA. We found that the peak strain and pseudo-dynamic modulus of leiomyoma tissue was significantly increased relative to matched myometrium. In addition, leiomyoma cells demonstrated an attenuated response to applied cyclic uniaxial strain and to variation in substrate stiffness, relative to myometrial cells. However, on a flexible pronectin-coated silicone substrate, basal levels and lysophosphatidic acid-stimulated levels of activated RhoA were similar between leiomyoma and myometrial cells. In contrast, leiomyoma cells plated on a rigid polystyrene substrate had elevated levels of active RhoA, compared to myometrial cells. The results indicate that viscoelastic properties of the ECM of leiomyoma contribute significantly to the tumor's inherent stiffness and that leiomyoma cells have an attenuated sensitivity to mechanical cues. The findings suggest there may be a fundamental alteration in the communication between the external mechanical environment (extracellular forces) and reorganization of the actin cytoskeleton mediated by RhoA in leiomyoma cells. Additional research will be needed to elucidate the mechanism(s) responsible for the attenuated mechanical signaling in leiomyoma cells.

Insurance mandates, embryo transfer, outcomes--the link is tenuous.

To examine the relationship between state insurance mandate status and the number of embryos transferred in assisted reproductive technology cycles, we conducted a retrospective analysis of clinics reporting to the publicly available national Society for Assisted Reproductive Technology registry. We found that clinics in states with comprehensive mandates transferred between 0.210 and 0.288 fewer embryos per cycle depending upon patient age, and were more likely to transfer fewer embryos than recommended for older women; however, the relationship between state mandate status and clinic birth and multiple birth rates varied by age group.

Why leiomyomas are called fibroids: the central role of extracellular matrix in symptomatic women.

Uterine leiomyomas are highly prevalent and symptomatic tumors of women in their reproductive years. The morbidity caused by these tumors is directly related to increasing size. Leiomyoma cells do not rapidly proliferate; instead, the tumors grow primarily due to excessive production of disorganized extracellular matrix (ECM). The aberrant ECM results from excessive production of collagen subtypes and proteoglycans, increased profibrotic cytokines including transforming growth factors beta1 and beta3, and decreased or disrupted matrix metalloproteinases. These alterations result in the development of an ECM that is exceptionally stable. As a result, therapeutic interventions must redirect leiomyoma cells toward extracellular matrix dissolution, rather than solely inhibiting cell proliferation. Gonadotropin-releasing hormone analogues and selective progesterone receptor modulators with demonstrated clinical efficacy provide such a change in abnormal extracellular matrix formation by leiomyoma cells, inhibiting and reversing the fibrotic process. Novel therapies using pathways distinct from gonadal hormones, including antifibrotics, retinoic acid, peroxisome-proliferator-activated receptor gamma ligands, and curcumin, provide promise for a future with improved therapeutic options for women suffering from uterine leiomyomas.

Use of F 18-fluoro-D-glucose-positron emission tomography-computed tomography to localize a hilar cell tumor of the ovary.

OBJECTIVE: To describe provocative testing and alternative imaging strategies used to localize an androgen-producing tumor in a 58-year-old woman with severe hirsutism. DESIGN: Case report. SETTING: Clinical Research Center. PATIENT(S): A 58-year-old woman who was seen for evaluation of severe hirsutism. INTERVENTION(S): Serum androgen levels were measured at baseline, 4 hours after administration of 2000 IU of hCG, and 11 days after administration of 3.75 mg of leuprolide acetate (LA). Magnetic resonance imaging and F 18-fluoro-D-glucose-positron emission tomography-computed tomography (FDG-PET/CT) were performed. MAIN OUTCOME MEASURE(S): Description of preoperative provocative testing and imaging. RESULT(S): In response to hCG, T rose from 243 to 288 ng/dL then decreased to 233 ng/dL after LA administration. The FDG-PET/CT scan demonstrated focal hypermetabolism in the right pelvis, corresponding to a soft-tissue density on the noncontrast CT scan. Magnetic resonance images were correlated with the PET/CT, and the right ovary was identified. Right salpingo-oophorectomy was performed, and final pathologic examination revealed a hilar cell tumor with ovarian cortical hyperplasia. CONCLUSION(S): This case demonstrates the utility of provocative testing in the evaluation of a patient with severe hirsutism and illustrates the value of FDG-PET/CT when traditional imaging is nondiagnostic.

Conversion from assisted reproductive technology to intrauterine insemination in low responders: is it advantageous?

OBJECTIVE: To examine cycle outcomes among patients demonstrating an attenuated ovarian response that proceeded to oocyte retrieval to those converted to intrauterine insemination (IUI). DESIGN: Retrospective cohort study. SETTING: Large private fertility center. PATIENT(S): First planned autologous assisted reproductive technology (ART) cycles among women demonstrating a poor ovarian response to hyperstimulation (≤4 follicles≥14 mm, peak E2<1,000 IU/L at hCG administration). INTERVENTION(S): Oocyte retrieval or IUI conversion. MAIN OUTCOME MEASURE(S): Live birth and clinical pregnancy. RESULT(S): A total of 269 IUI conversions and 167 oocyte retrievals followed a poor ovarian response to gonadotropins among first planned ART cycles. Number of follicles≥14 mm (2.3 vs. 3.5) and peak E2 levels (555 vs. 743 pg/mL) were lower for IUI conversions compared with those proceeding to ART. Peak E2 was similar between groups after adjusting for follicle number (IUI: 611 pg/mL; ART: 652 pg/mL). Stimulation response was similar between treatment groups with equivalent follicle numbers. Undergoing oocyte retrieval was associated with significantly improved pregnancy (odds ratio [OR] 3.6, 95% confidence interval [CI] 1.8-7.4) and live birth outcome (OR 3.5, 95% CI 1.7-8.0) after adjusting for age and follicle number. CONCLUSION(S): Among women demonstrating a poor ovarian response to gonadotropins, proceeding with planned ART resulted in significantly higher pregnancy rates than converting these cycles to IUI.

Transforming growth factor beta3 regulates the versican variants in the extracellular matrix-rich uterine leiomyomas.

Uterine leiomyoma are common, benign tumors that are enriched in extracellular matrix. The tumors are characterized by a disoriented and loosely packed collagen fibril structure similar to other diseases with disrupted Transforming growth factor beta (TGF-beta) signaling. Here we characterized TGF-beta3 signaling and the expression patterns of the critical extracellular matrix component versican in leiomyoma and myometrial tissue and cell culture. We also demonstrate the regulation of the versican variants by TGF-beta3. Using leiomyoma and matched myometrium from 15 patients, messenger RNA (mRNA) from leiomyoma and myometrium was analyzed by semiquantitative real time reverse transcription-polymerase chain reaction (RT-PCR), while protein analysis was done by western blot. Transforming growth factor beta3 transcripts were increased 4-fold in leiomyoma versus matched myometrium. Phosphorylated-TGF-beta RII and phosphorylated-Smad 2/3 complex were greater in leiomyoma as documented by Western blot. The inhibitor Smad7 transcripts were decreased 0.44-fold. The glycosaminoglycan (GAG)-rich versican variants were elevated in leiomyoma versus myometrial tissue: specifically V0 (4.27 +/- 1.12) and V1 (2.01 +/- 0.27). Treatment of leiomyoma and myometrial cells with TGF-beta3 increased GAG-rich versican variant expression 7 to 12 fold. Neutralizing TGF-beta3 antibody decreased the expression of the GAG-rich versican variants 2 to 8 fold in leiomyoma cells. Taken together, the aberrant production of excessive and disorganized extracellular matrix that defines the leiomyoma phenotype involves the activation of the TGF-beta signaling pathway and excessive production of GAG-rich versican variants.

Management of uterine fibroids in the patient pursuing assisted reproductive technologies.

Uterine leiomyomas are present in 30-70% of women of reproductive age. In addition to causing menstrual disorders and pain, uterine fibroids negatively affect fertility and pregnancy outcome for patients pursuing assisted reproduction. The two questions that have to be addressed are: which fibroids should be treated and how should they be treated? Submucosal fibroids are associated with a 70% reduction in delivery rate. Intramural fibroids had a lesser effect and reduced the delivery rate by approximately 30%. By contrast, studies have demonstrated that subserosal fibroids did not negatively impact fertility. Furthermore, both submucosal and intramural fibroids were associated with an increased risk of spontaneous miscarriage. Myomectomy is considered the treatment of choice to alleviate these detrimental effects. Further research is needed before alternative treatments can be recommended.

Low serum osteoprotegerin levels in premenopausal infertile women with low bone density: an association of relevance?

OBJECTIVE: To evaluate if the levels of the central regulators of osteoclast activity, osteoprotegerin (OPG) and receptor activator for nuclear factor kappaB-ligand (RANk-L), correlate with bone mineral density (BMD) parameters in premenopausal infertile women. DESIGN: Cross-sectional study. SETTING: Academic infertility practice. PATIENT(S): 82 Infertile, but otherwise healthy, premenopausal women< 42 years age presenting for routine care. INTERVENTION(S): BMD assessment; serum samples (cycle days 1 to 3), questionnaire addressing demographics, medical, social, family, and personal histories. MAIN OUTCOME MEASURE(S): BMD categorized as "Low" (Z -1.0); Biomarkers of bone metabolism (BAP, TRAP, NTX, OPG, RANK-L) and ovarian reserve (FSH, estradiol). RESULT(S): The prevalence of low BMD was 23% (19/82). Significantly lower levels of OPG were observed in association with low BMD compared with normal BMD (1.37 +/- 0.57 versus 2.0 +/- 1.05 pMol/L). Multivariable logistic regression analyses revealed low OPG levels as independent predictors of low BMD after adjusting for parameters known to influence bone density. CONCLUSION(S): We demonstrate an independent relationship between low circulating levels of OPG and low BMD in premenopausal healthy, albeit infertile, women.

Labial fusion: a rare complication of chronic graft-versus-host disease.

BACKGROUND: Hematopoetic stem cell transplantation may be complicated by genital graft-versus-host (GVH) disease and may uncommonly result in labial fusion. CASE: A 22-year-old woman with a history of Ewing's sarcoma and acute myelogenous leukemia received chemotherapy and total-body irradiation followed by a matched, unrelated donor hematopoetic stem cell transplantation. After transplantation, she developed chronic GVH disease involving the skin, eyes, mouth, and joints. Concomitant vulvar pruritus was presumed to be a fungal infection but, in fact, was genital GVH disease manifesting as labial fusion. Topical estrogen, topical steroids, and surgical division of the labia were successful. CONCLUSION: Genital GVH disease should be considered in women with genital tract complaints after hematopoetic stem cell transplantation. Labial fusion secondary to chronic GVH disease may be treated successfully with surgery and medical therapy.

Mechanical homeostasis is altered in uterine leiomyoma.

OBJECTIVE: Uterine leiomyoma produce an extracellular matrix (ECM) that is abnormal in its volume, content, and structure. Alterations in ECM can modify mechanical stress on cells and lead to activation of Rho-dependent signaling and cell growth. Here we sought to determine whether the altered ECM that is produced by leiomyoma was accompanied by an altered state of mechanical homeostasis. STUDY DESIGN: We measured the mechanical response of paired leiomyoma and myometrial samples and performed immunogold, confocal microscopy, and immunohistochemical analyses. RESULTS: Leiomyoma were significantly stiffer than matched myometrium. The increased stiffness was accompanied by alteration of the ECM, cell shape, and cytoskeleton in leiomyoma, compared with myometrial samples from the same uterus. Levels of AKAP13, a protein that is known to activate Rho, were increased in leiomyoma compared to myometrium. AKAP13 was associated with cytoskeletal filaments of immortalized leiomyoma cells. CONCLUSION: Leiomyoma cells are exposed to increased mechanical loading and show structural and biochemical features that are consistent with the activation of solid-state signaling.

Vasomotor symptoms in infertile premenopausal women: a hitherto unappreciated risk for low bone mineral density.

OBJECTIVE: To identify the prevalence of vasomotor symptoms (VMS) in a population of premenopausal infertile women and to determine whether VMS is associated with enhanced bone turnover and low bone mineral density (BMD). DESIGN: Cross-sectional study. SETTING: Academic infertility practice. PATIENT(S): Eighty-two premenopausal infertile, but otherwise healthy, women attending for routine infertility care. INTERVENTION(S): Bone mineral density testing, general health and Profile of Mood States questionnaires, and serum samples (cycle d 1-3). MAIN OUTCOME MEASURE(S): Vasomotor symptoms, specifically hot flashes (HF) and night sweats (NS); BMD z score, BMD categorized as low (Z -1.0); ovarian reserve assessment (biochemical and ovarian dimensions on transvaginal ultrasound); and serum markers of bone turnover (collagen N-terminal telo-peptide, tartrate-resistant acid phosphatase, and bone-specific alkaline phosphatase) and ovarian reserve (FSH, E(2), and inhibin B). Multivariable regression analyses determined the associations between VMS, BMD, and bone turnover (individual markers and composite turnover score). RESULT(S): The prevalence of VMS was 12% in this relatively young population (mean [+/- SD] age [years], 34.53 +/- 4.32). Symptomatic women were statistically significantly more likely to report sleep disturbances and to exhibit evidence of low BMD, as well as to exhibit enhanced bone turnover and poorer ovarian reserve parameters. Multivariable logistic regression analyses confirmed the statistical significance of both HF and NS as independent correlates to low BMD after adjusting for age, body mass index, smoking status, menstrual regularity, and ovarian reserve status. Multivariable linear regression analyses demonstrated that NS, but not HF, predicted higher bone turnover at a statistically significant level after adjusting for age, smoking, menstrual regularity, and ovarian reserve. CONCLUSION(S): We demonstrate, in a premenopausal population of infertile women, evidence of morbid accompaniments to VMS, including sleep disturbances and evidence of low BMD. Our data further suggest a state of enhanced bone turnover in association with VMS, specifically in those experiencing NS. Declining ovarian reserve may be the common pathophysiological mechanism underlying VMS and low BMD in the symptomatic population and merits further investigation.

Simple tools for assessment of ovarian reserve (OR): individual ovarian dimensions are reliable predictors of OR.

OBJECTIVE: To determine the association between individual ovarian dimensions, advancing age, and declining ovarian reserve (OR) in an infertile population. DESIGN: An ongoing prospective observational study. SETTING: Academic infertility practice. PATIENT(S): Sixty-nine premenopausal women presenting for the evaluation and management of infertility. INTERVENTION(S): Transvaginal ultrasound assessment of the ovarian dimensions (length, width, and overall diameter in cm) and evaluation of the OR status by measuring serum levels of FSH and E(2) in the early follicular phase of the menstrual cycle. MAIN OUTCOME MEASURE(S): Relationship of the individual ovarian dimensions to age (years) and OR (reflected by historical maximal FSH levels). RESULT(S): A statistically significant decrease in three ovarian size parameters accompanied advancing age (ovarian width, r = -0.30; ovarian length, r = -0.24; and the mean overall ovarian diameter, r = -0.30). Levels of FSH demonstrated a significant and linear correlation with age (r = 0.39). Increasing levels of FSH (and hence declining OR) were associated with statistically significant declines in the mean ovarian width (r = -0.39), length (r = -0.38), and overall mean ovarian diameter (r = -.42), Patients with a known diagnosis of diminished OR demonstrated significantly reduced ovarian dimensions compared with patients with other infertility etiologies. Multivariate linear regression analysis confirmed individual ovarian measurements (width, length, and overall diameter) as independent predictors of OR (FSH levels) after adjusting for parameters that are known to influence ovarian size, i.e., age, smoking status, body mass index, and anovulation history. Ovarian width emerged as the strongest predictor of OR (largest beta coefficient and hence the strongest association with OR status), compared with ovarian length and the mean ovarian diameter. CONCLUSION(S): Single ovarian dimensions are reliable predictors of advancing age and declining OR status in premenopausal infertile women. The magnitude of this association is most robust for the ovarian width.

Total knee arthroplasty in hemophilic arthropathy.

BACKGROUND: Arthropathy of the knee frequently develops in patients with hemophilia, who may require a total knee arthroplasty at a young age. Hemophilic patients, who require regular intravenous replacement of coagulation factor, have a higher prevalence of human immunodeficiency virus (HIV) infection, which can compromise the outcome of the arthroplasty. The purpose of this study was to evaluate prosthetic survival following total knee arthroplasty and identify factors associated with failures of the arthroplasties in hemophilic patients. METHODS: The results of fifty-three total knee arthroplasties performed in thirty-eight patients (twenty-nine of whom were seropositive for HIV) to treat hemophilic arthropathy between 1976 and 1998 were retrospectively reviewed. Inpatient and outpatient medical records were studied to determine the HIV status, CD4 lymphocyte count, type of prosthesis, duration of prosthetic survival, cause of failure, and cause of death. If an arthroplasty failed, the outcome of the treatment of the failed arthroplasty was also determined. RESULTS: The rate of survival of the prostheses was 90% after five years. Eleven total knee arthroplasties failed. The most common cause of failure was infection (seven knees), which developed at an average of sixty months (range, three to 138 months) after the arthroplasty. There was no significant difference in the CD4 lymphocyte counts between the patients in whom infection developed and those in whom it did not. The HIV status also did not appear to be related to the development of infection. Thirteen patients died, and the most common cause of death was complications associated with acquired immunodeficiency syndrome (AIDS). CONCLUSIONS: Total knee arthroplasty performed to treat hemophilic arthropathy has a high risk of failure as a result of infection. Most infections developed late and were frequently caused by Staphylococcus epidermidis, suggesting that a likely cause of failure due to infection was hematogenous spread during administration of coagulation factor. It may be difficult to salvage a prosthesis complicated by infection. However, the life expectancy of hemophilic patients is lower than that of the general population of patients treated with total knee arthroplasty, and the improvement in the quality of life after total knee arthroplasty for hemophilic arthropathy may outweigh the risk of failure.