The combined presence of CD20 + B cells and PD-L1 + tumor-infiltrating lymphocytes in inflammatory breast cancer is prognostic of improved patient outcome.

PURPOSE: The purpose of the study was to evaluate protein expression of PD-L1 and CD20 as prognostic biomarkers of patient outcome in inflammatory breast cancer (IBC) samples. METHODS: PD-L1 and CD20 protein expression was measured by immunohistochemistry in 221 pretreatment IBC biopsies. PD-L1 was assessed in tumor cells (PD-L1+ tumor cells) and tumor stromal infiltrating lymphocytes (PD-L1+ TILs); CD20 was scored in tumor-infiltrating B cells. Kaplan-Meier curves and Cox proportional hazard models were used for survival analysis. RESULTS: PD-L1+ tumor cells, PD-L1+ TILs, and CD20+ TILs were found in 8%, 66%, and 62% of IBC, respectively. PD-L1+ tumor cells strongly correlated with high TILs, pathological complete response (pCR), CD20+ TILs, but marginally with breast cancer-specific survival (BCSS, P = 0.057). PD-L1+ TILs strongly correlated with high TILs, CD20+ TILs, and longer disease-free survival (DFS) in all IBC and in triple-negative (TN) IBC (P < 0.035). IBC and TN IBC patients with tumors containing both CD20+ TILs and PD-L1+ TILs (CD20+TILs/PD-L1+TILs) showed longer DFS and improved BCSS (P < 0.002) than patients lacking both, or those with either CD20+ TILs or PD-L1+ TILs alone. In multivariate analyses, CD20+TILs/PD-L1+TILs status was an independent prognostic factor for DFS in IBC (hazard ratio (HR): 0.53, 95% CI 0.37-0.77) and TN IBC (HR: 0.39 95% CI 0.17-0.88), and for BCSS in IBC (HR: 0.60 95% CI 0.43-0.85) and TN IBC (HR: 0.38 95% CI 0.17-0.83). CONCLUSION: CD20+TILs/PD-L1+TILs status represents an independent favorable prognostic factor in IBC and TN IBC, suggesting a critical role for B cells in antitumor immune responses. Anti-PD-1/PD-L1 and B cell-activating immunotherapies should be explored in these settings.

The joint contribution of tumor phenotype and education to breast cancer survival disparity between Hispanic and non-Hispanic white women.

Some studies suggest that Hispanic women are more likely to have ER- and triple-negative (ER-/PR-/HER2-) tumors and subsequently poorer prognosis than non-Hispanic white (NHW) women. In addition, only a handful of studies have examined period-specific effects of tumor phenotype and ethnicity on breast cancer survival, leaving the time-varying effects of hormonal status and ethnicity on breast cancer survival poorly defined. This study describes short and long-term breast cancer survival by ethnicity at 0-5 years and 5+ years post-diagnosis using data from the New Mexico Health, Eating, Activity, and Lifestyle cohort of Hispanic and NHW women ages 29-88 years newly diagnosed with stages I-IIIA breast cancer. The survival rate for Hispanics at 0-5 years was 82.2 % versus 94.3 % for NHW. Hispanics were more likely to have larger tumors, more advanced stage, and ER- phenotypes compared to NHW women. There was a significantly higher risk of breast cancer mortality in Hispanics over 5 years of follow-up compared to NHW (HR = 2.78, 95 % CI 1.39-5.56), adjusting for age, tumor phenotype, stage, and tumor size. This ethnic difference in survival, however, was attenuated and no longer statistically significant when additional adjustment was made for education, although a >1.5-fold increase in mortality was observed. In contrast, there was no difference between ethnic groups for survival after 5 years (HR = 1.08, 95 % CI 0.36-3.24). Our results indicate that the difference in survival between Hispanic and NHW women with breast cancer occurs in the first few years following diagnosis and is jointly associated with tumor phenotype and socio-demographic factors related to education.

Epidermal growth factor receptor as a biomarker for cervical cancer.

This review focuses on the different modes of expression of the epidermal growth factor receptor (EGFR). All methods used to assess EGFR expression are critically analyzed and insights into the use of inhibitors of EGFR for treatment of cervical cancer are discussed. Currently, expression of EGFR as a biomarker for prognosis or for treatment of cervical cancer is not defined for clinical use.

Absence of epidermal growth factor receptor mutations in cervical cancer.

Epidermal growth factor receptor (EGFR) is overexpressed in the majority of cervical cancers (CCs). Somatic mutations of EGFR have been associated with clinical response to tyrosine kinase inhibitors (TKIs) in lung cancer patients. This study was designed to establish the frequency of EGFR point mutations in patients diagnosed with high-grade squamous intraepithelial lesions (HSIL) and CC. Nine cell lines derived from CC were screened for EGFR mutations in exons 18 through 21. Eighty-nine patient samples derived from invasive CC (n = 80) and HSIL (n = 9) were analyzed for the presence of EGFR mutations in exons 19 and 21. We found no mutations affecting the EGFR kinase domain in exons 18 through 21 in all cell lines tested, and no EGFR mutations were detected in exons 19 and 21 in all 89 human neoplastic samples analyzed. These data indicate that mutations in the EGFR kinase domain are very rare in CC and HSIL. Our results suggest, therefore, that treatment of CC patients with TKIs may not have the same efficacy as seen in patients with lung cancer, and that targeting the EGFR with other inhibitors may be more appropriate.

Fine needle aspiration cytology of follicular dendritic cell sarcoma. Report of a case with cytologic detection in an extranodal site.

BACKGROUND: Follicular dendritic cell sarcoma (FDCS) is a rare malignancy of accessory immune cells that can present in both nodal and extranodal sites. Previous cytologic case reports of FDCS have focused on fine needle aspiration (FNA) findings in nodal sites with low grade morphology and indolent clinical courses. CASE: A 33-year-old female presented with a three-month history of abdominal distention, early satiety and nausea. Initial imaging studies showed a large abdominal mass, with subsequent studies showing lung, liver and lymph node metastases. Examination of primary and metastatic tumors by a combination of conventional histology, immunohistochemistry and FNA demonstrated an extranodal intraabdominal follicular dendritic cell sarcoma. CONCLUSION: FDCS demonstrates a characteristic cytologic picture on FNA, with important cytologic features, including both syncytial and discohesive large epithelioid to spindled malignant cells with intranuclear inclusions, nuclear grooves and a prominent, mature, lymphocytic inflammatory component. No evidence of morphologic tumor progression was noted in comparison of primary and metastatic tumors. To aid in the cytologic distinction of FDCS from other similar-appearing neoplasms, we recommend acquisition of material for immunohistochemical studies, recognition of diverse clinical presentations (including extranodal and aggressive) and acknowledgment of the range of tumor morphologic grades.

Patient and tumor characteristics of colon cancers with microsatellite instability: a population-based study.

Molecular screening for microsatellite instability (MSI) in colon cancers has been proposed to identify individuals with hereditary nonpolyposis colorectal cancer. To date, most reports of MSI in colorectal cancer have been based on studies of clinical case series or high-risk families. We examined the proportion of incident colon cancers in the general population that exhibit MSI by patient and tumor characteristics. We interviewed 201 colon cancer cases ascertained by the New Mexico Tumor Registry in the metropolitan Albuquerque area for demographic information, lifestyle factors, medical history, and family cancer history. Paired normal and tumor tissue specimens were obtained for each case. Three microsatellite markers were used; instability was defined as observed alteration at two or more loci. Overall, 37 of 201 (18%) colon cancers exhibited instability. MSI was more common among cases >70 years (26%) and most common among cases >80 years (38%). MSI was significantly associated with tumors in the proximal colon and with later stage and poor differentiation among cases >70 years. MSI was not associated with a history of polyps. Family history of colorectal cancer was associated with MSI only among cases <50 years. When all factors were analyzed jointly in a regression model, proximal subsite and poor differentiation remained significantly associated with MSI. One patient, whose tumor exhibited MSI, fulfilled the Amsterdam Criteria for hereditary nonpolyposis colorectal cancer. Our study provides a population-based estimate of MSI in colon tumors and a representative estimate of the proportion of colorectal cancer patients in the general population who consent to be interviewed for family cancer history and to have biological samples analyzed.

The lingula is an appropriate site for lung biopsy.

BACKGROUND: Lung biopsy is commonly performed for diagnosis of diffuse pulmonary disease. The lingula offers technical advantages for biopsy, however the quality of tissue obtained by lingula biopsy has been questioned. We sought to determine whether lingula biopsy was a satisfactory site for biopsy in terms of diagnostic yield, therapeutic interventions, and survival results. METHODS: All diagnostic lung biopsies performed for diffuse lung disease at 3 university affiliated hospitals between July 1, 1992 and December 31, 1998 were retrospectively reviewed. Patients were divided into 2 groups, depending upon site of biopsy: patients with lingula biopsy only and those with biopsies from other sites. RESULTS: There were 75 patients; 20 underwent biopsy of the lingula alone, 48 had biopsy of other sites with or without biopsy of the lingula, and location of biopsy was unknown in 7 patients. Histologic diagnosis was achieved in all patients. Significant beneficial therapeutic changes were made in 14 lingula patients, and consisted of immunosuppression in 12 cases. Three patients died in the hospital or within 30 days. Fourteen patients survived 1 year. There was no significant difference between patients that had biopsy of the lingula alone and those that had biopsies from other sites in urgency, technique, histologic diagnosis, rate of therapeutic interventions, hospital mortality, or 1 year survival. CONCLUSIONS: Lung biopsy of the lingula compared to other anatomic sites has equivalent diagnostic yield, therapeutic significance, and survival. Given the technical ease of biopsy, when disease is present radiographically it is the preferred site for lung biopsy.

Biologic characteristics of interval and screen-detected breast cancers.

BACKGROUND: Interval breast cancer is defined as a cancer that is detected within 12 months after a negative mammogram. The failure of mammography to detect breast cancer depends on testing procedures, radiologist interpretation, patient characteristics, and tumor properties. Although errors by radiologists explain some interval cancers, another explanation is that the tumor is rapidly growing and was too small to be detected on the last mammogram. To determine whether markers of tumor growth rate are associated with risk of an interval cancer, we conducted a population-based study with the use of data collected statewide by the New Mexico Mammography Project. METHODS: Among women who received a mammographic examination from 1991 throughout 1993, we ascertained records of all patients with breast cancer diagnosed within 12 months of a negative screening mammographic examination (interval cancers) and corresponding tumor samples, when available. We selected an age- and ethnicity-matched comparison group of control patients with screen-detected breast cancers diagnosed during the same period. In tumor samples, p53, bcl-2, and Ki-67 were examined immunologically and the apoptotic index was assessed histologically. We used logistic regression to determine whether interval cancers were associated with selected demographic, radiologic, and biologic characteristics. RESULTS: It is more likely that mammography did not detect tumors with a high proportion of proliferating cells (>20%) than tumors with a low proportion of proliferating cells (<5%) (odds ratio [OR] = 4.09; 95% confidence interval [CI] = 1.14-14.65). The OR for mammographic failure was 2.96 (95% CI = 1.07-8.20) among cancers that expressed p53 compared with cancers that did not. Interval cancers also had fewer apoptotic cells. Approximately 75% of interval cancers appear to have tumors with 5% proliferating cells or more. Younger women had a higher proportion of rapidly proliferating and aggressive cancers. CONCLUSION: Rapidly growing and aggressive tumors account for a substantial proportion of mammographic failure to detect breast cancer, especially among younger women, who have a high proportion of aggressive cancers.

Lung biopsy: is it necessary?

OBJECTIVE: Lung biopsy is associated with substantial mortality rates. We reviewed our experience with this operation, primarily in patients with immunocompetence, to determine whether the results justify the continued performance of this procedure. METHODS: We conducted a retrospective review of all diagnostic lung biopsies performed at 3 university-affiliated hospitals between July 1, 1992, and December 31, 1998. RESULTS: There were 75 patients: 25 patients were treated electively, 17 were treated on an urgent basis, 27 patients on an emergency basis, and the urgency was unclear in 6 patients. Significant beneficial therapeutic changes were made in 15 of 25 elective procedures (60%), in 16 of 17 urgent procedures (94%), and in 11 of 27 emergency procedures (41%; P =.001). Significant beneficial therapeutic changes consisted of immunosuppression in 13 of 15 (87%) patients treated on an elective basis, in 9 of 16 (56%) treated on an urgent basis, and in 9 of 11 (82%) treated on an emergency basis in whom therapy was altered (P =.14). Operative death was 0 of 25 for elective operations (0%), 3 of 17 for urgent operations (18%), and 14 of 26 for emergency operations (54%). Multivariable analysis of operative death showed urgency to be the only significant predictor of death (P =.002). CONCLUSIONS: In patients with immunocompetence, elective and urgent lung biopsies have acceptable operative mortality rates and frequently result in important beneficial therapeutic changes. Consequently biopsies are appropriate in these patients. Emergency biopsies are associated with high operative mortality rates and rarely result in a therapeutic change other than immunosuppression. These patients should not undergo lung biopsy if they are in stable condition and should be treated empirically with immunosuppression without operation if their condition is deteriorating.

Association between outcome and telomere DNA content in prostate cancer.

PURPOSE: To perform an initial retrospective investigation of the relationship between outcome in patients with organ confined prostate adenocarcinoma and the tumor cells' content of telomere DNA. MATERIALS AND METHODS: The case-controlled study group was composed of eighteen men diagnosed with prostatic adenocarcinoma prior to 1993. The group was selected so that approximately one half died within ten years of diagnosis and one half survived ten years or longer. Archival, paraffin-embedded tumor tissue was recovered for each patient. DNA was extracted from newly cut sections, fixed to nylon membranes and hybridized with P32-labeled centromere- and telomere-specific probes. Telomere DNA contents were quantitated from the hybridized radioactivities. The relationships between telomere DNA content and survival, and telomere DNA content and disease recurrence in men receiving prostatectomies were determined. RESULTS: Death and disease recurrence were associated with reduced telomere DNA content (p <0.0001, p <0.0001, respectively). CONCLUSIONS: Telomere DNA content may differentiate high-risk patients with metastatic prostate cancer from men with indolent disease who can be spared the unnecessary side effects and expense of treatment by management with "watchful waiting."

Fine needle aspiration cytology of plexiform fibrohistiocytic tumor. A case report.

BACKGROUND: Plexiform fibrohistiocytic tumors are rare lesions of proposed myofibroblastic origin occurring primarily in infants and children. While the histologic, immunohistochemical and ultrastructural findings have been well described, cytologic description has been limited. CASE: An 8-month-old, male infant presented with a posterior chest wall mass and decreased use of his left arm. Fine needle aspiration biopsy showed a spectrum of plump fibroblastic spindle cells and histiocytelike cells within a finely granular myxoid background. Osteoclastlike giant cells were also noted. CONCLUSION: We report here the cytologic findings of a plexiform fibrohistiocytic tumor from fine needle aspiration biopsy studied using Papanicolaou, Ultrafast Papanicolaou and Diff-Quik stain, with the cytologic differential diagnosis of other spindled and histiocytelike tumors.

Reduced telomere DNA content is correlated with genomic instability and metastasis in invasive human breast carcinoma.

Telomere shortening leads to genomic instability and has been correlated with poor outcome in several types of cancer. A recently described, robust titration assay was used to quantify telomere DNA content in frozen and paraffin-embedded specimens of 49 invasive human breast carcinomas, including tumors with normal or abnormal contents of genomic DNA, which produced regional, distant, or local disease. Telomere DNA contents ranged from 53% to 370% of the content in a reference DNA purified from normal placenta. Tumors were divided into three groups of approximately equal size based on increasing telomere DNA content. All of 16 tumors in the group with the least telomere DNA (Group I), were aneuploid compared to 9/17 tumors in the group with the most telomere DNA (Group III). The Chi-square test for trend indicated that tumors with the least telomere DNA were significantly more likely to be aneuploid than tumors with the most telomere DNA (p < 0.002). Twelve of 14 tumors in Group I also produced metastatic disease compared to 8/15 tumors in Group III. The Fischer Exact Test indicated that tumors with the least telomere DNA were significantly more likely to be metastatic than tumors with the most telomere DNA (p < 0.05). There was no association between telomere DNA content and patients' age, tumors' size, grade, stage, or fraction of cells in S-phase. The correlation of reduced telomere DNA content with aneuploidy and metastasis, both of which are associated with poor outcome in invasive breast carcinoma, implies that telomere DNA content also could have prognostic value.

Cytologic detection of microsporidia spores in bile. A comparison of stains.

OBJECTIVE: To compare stains in preparations of bile in a patient with AIDS and microsporidial cholangitis. STUDY DESIGN: Bile was obtained from a 30-year-old male with AIDS and symptoms of cholangitis. Comparative staining of the specimen was performed using a formalin-fixed preparation stained with Chromotrope 2R stain and with alcohol-fixed preparations stained with Gram and Giemsa stain and Diff-Quik. An alcohol-fixed ThinPrep slide was stained with Papanicolaou stain. RESULTS: Diagnostic microsporidia spores were detected under oil immersion using Papanicolaou, Chromotrope 2R, Giemsa and Gram stain. The Diff-Quik-stained preparation also revealed microsporidia but with suboptimal morphology. CONCLUSION: Detection of microsporidia in bile can be achieved using several different stains routinely available to cytologists, most optimally with alcohol-fixed Papanicolaou- or Giemsa-stained preparations or with Chromotrope 2R stain, which is available in parasitology laboratories. These findings should be applicable to fluids from other body sites with this emerging pathogen in AIDS.

Plexiform fibrohistiocytic tumor with a clonal cytogenetic anomaly.

Plexiform fibrohistiocytic tumors are rare lesions of proposed myofibroblastic origin occurring primarily in infants and children. There is a characteristic biphasic histology comprised of both fibroblastic and histiocyte-like components. These tumors tend to be locally aggressive with prognosis dependent on completeness of resection. A previous cytogenetic case report of this tumor described a stemline clone with a karyotype of 46,XY,-6,-8, del(4)(q25q31),del(20)(q11.2),+der(8)t(8;?) (p22;?),+mar. We report a different cytogenetic finding in another plexiform fibrohistiocytic tumor which demonstrated a simpler karyotype of 46,XY,t(4;15)(q21;q15). The implications of cytogenetic heterogeneity in fibroblastic tumors is briefly discussed.

Bethesda classification of cervicovaginal smears: reproducibility and viral correlates.

Fifty-five cervicovaginal smears from women with squamous intraepithelial lesions (SILs) were independently evaluated on two separate occasions by four cytopathologists using a binary classification system (the Bethesda system). Smears were categorized as low-grade (LSIL) or high-grade (HSIL) using previously published criteria. All women had subsequent cervical biopsies containing human papillomavirus (HPV) DNA amplified with the polymerase chain reaction and typed by restriction fragment polymorphism analysis. Three or more observers agreed on classification in 49 of 55 cases (87%); unanimous diagnoses were rendered in 31 cases (56%). Interobserver and intraobserver reproducibility ranged from fair to near-excellent (kappa values 0.40 to 0.63; 0.63 to 0.74, respectively). HPV types included HPV 16 (27%), 18 (7%), 31 (9%), 35 (4%), 39 (4%), 6 (10%), 11 (2%), novel types (30%), and multiple types (4%). High-risk HPV types (16, 18, 31, 35, and 39) were significantly associated (P = .03) with consensus HSIL diagnoses (agreement of three or more observers). This was primarily because of the strong association of HPV 16 with HSIL (P = .001). After excluding HPV 16, the other high-risk HPV types (18, 31, 35, and 39) were no longer significantly associated with consensus HSIL diagnoses (P > .5). Conversely, LSIL diagnoses were significantly associated with non-high-risk HPV types (all HPV types except 16, 18, 31, 35, and 39; P = .006). Binary cytological classification of cervicovaginal SILs is reproducible among cytopathologists. Such classification correlates well with most low-risk HPV types and with the prototypic high-risk HPV 16 but not with other high-risk HPV types.

Autopsy verification of Encephalitozoon intestinalis (microsporidiosis) eradication following albendazole therapy.

Microsporidian infections are increasingly recognized as an important cause of morbidity for persons infected with the human immunodeficiency virus. Encephalitozoon (formerly Septata) intestinalis is a recently described microsporidian that causes intestinal and disseminated infections in severely immunocompromised patients with acquired immunodeficiency syndrome. Several studies suggest that albendazole is an effective therapy for E intestinalis infection. However, relapses of symptoms and reappearance of microsporidian spores in diagnostic specimens have been reported following treatment in some cases. Because these results are based on examination of feces or cytologic specimens with an inherent sampling bias, it would be ideal to have autopsy data on the complete tissue evaluation of major organ systems of patients who had antemortem E intestinalis infection treated with albendazole. This report describes an acquired immunodeficiency syndrome patient with diarrhea and wasting syndrome associated with E intestinalis infection. Treatment with albendazole produced relief of his clinical symptoms and eliminated microsporidian spores in his feces. Following his death from other causes, an autopsy was performed. Comprehensive microscopic examination of all major organs revealed no evidence of residual microsporidian infection, suggesting parasitologic cure of E intestinalis with albendazole. The postmortem finding of complete clearance of microsporidia from body tissues is significant for future albendazole treatment of patients infected with E intestinalis and provides strong support for the value of the autopsy in evaluating the therapeutic efficacy of antimicrobials in emerging infections.

Juvenile granulosa cell tumor of the infantile testis. Evidence of a dual epithelial-smooth muscle differentiation.

We report the ultrastructure and immunohistochemical profile of seven juvenile granulosa cell tumors of the infantile testis. The infants' ages ranged from 1 day to 11 months. All tumors had characteristics ultrastructure with a mixture of spindle smooth-muscle and theca cells and polygonal granulosa cells. Clusters of polygonal granulosa cells were invested by a continuous basal lamina and contained bundles of distinct cytoplasmic filaments with evenly distributed dense bodies resembling smooth muscle. These filaments were occasionally attached to well-developed, prominent desmosomes. Tumor cells had a conspicuous rough endoplasmic reticulum and Golgi complex and occasional neutral fat droplets. In all tumors, mitochondria had laminated cristae and only rarely were there cristae with a tubulovesicular pattern characteristic of steroid secreting cells. Tumor cells stained focally with low-molecular-weight cytokeratins (8,18, and 19), smooth-muscle-specific actin, desmin, and more noticeably with vimentin. These ultrastructural and immunohistochemical features of dual epithelial-mesenchymal differentiation and distinct muscle-like filaments with dense bodies are characteristic of the juvenile granulosa cell tumor of the infantile testis.

Cytologic/histologic correlation for quality control in cervicovaginal cytology. Experience with 1,582 paired cases.

For quality-control purposes, federal regulations require cytology laboratories to compare Papanicolaou smear and cervical biopsy reports, if available, and determine the cause of any discrepancies. The authors reviewed 56,497 cervicovaginal smears, of which 1,582 (2.8%) had a subsequent cervical biopsy or endocervical curettage within 2 months. A total of 175 discrepancies (11%) were identifed, and biopsies and smears from these cases were reviewed at a weekly conference. In the majority of cases, the diagnosis of the smear and biopsy was confirmed on review, and the discrepancy was attributed to sampling error (n = 159; 91%). Six cases (3.4% of all discrepant cases) were errors in cytologic diagnosis. Five of these were interpretation errors, and one case was a screening error. There were 10 errors in the evaluation of cervical biopsies (5.7% of all discrepant cases): five biopsies were undercalled, and five were overdiagnosed as a squamous intraepithelial lesion. The results of testing for human papillomavirus DNA by in situ hybridization were helpful in arbitrating some diagnositic disagreements.

Histology and Ureaplasma urealyticum culture in 63 cases of first trimester abortion.

The association between chorioamnionitis caused by Ureaplasma urealyticum and preterm delivery has been well documented. In contrast, a pathogenic role for Ureaplasma has been postulated in early spontaneous abortions, but definitive evidence for this association has been lacking. In 42 early spontaneous abortions and 21 elective abortions (both first trimester), the chorion was cultured for Ureaplasma. Each case was evaluated histologically for four features of inflammation and one feature of degeneration. For comparison, 32 selected placentas from third trimester preterm deliveries (11 with positive Ureaplasma cultures) were examined histologically for umbilical vasculitis and acute chorioamnionitis. In abortion specimens, Ureaplasma cultures were positive in 11 of 42 early spontaneous abortions (26%) versus 0 of 21 elective abortions (EABs). None of the five histologic features correlated with positive Ureaplasma cultures in early spontaneous abortions. In contrast, in preterm placentas, umbilical vasculitis, and acute chorioamnionitis correlated strongly with positive Ureaplasma cultures. The authors conclude that in premature delivery, U urealyticum chorionic culture positivity is associated with histologic chorioamnionitis; and in abortions, Ureaplasma chorionic culture positivity correlates with early spontaneous abortions (vs elective abortions), but not with histologic inflammation. This suggests that mechanisms of Ureaplasma pathogenesis other than acute inflammation should be considered in future studies of early spontaneous abortions.