Age Specific Risks of Uterine Cancer in Type 2 Diabetes and Associated Comorbidities in Taiwan.

Introduction: The global incidence of uterine cancer has increased substantially in recent decades. We evaluated if the trend of increasing prevalence of diabetes mellitus (DM) and obesity are attributed to the development of uterine cancer. Methods: Using data derived from the National Health Insurance database and Taiwan Cancer Registry, multivariate Cox proportional hazards regression models were adapted to analyze the risk factors of uterine cancer with potential confounding variables. Results: There were a total of 5,104,242 women aged 30−70 years enrolled in the study and 147,772 of them were diagnosed with DM during 2005−2007. In a total of 11 years of follow-up, 14,398 subjects were diagnosed with uterine cancer. An elevated risk of uterine cancer was observed in women with DM of all ages (HR 1.66, 95% CI 1.53−1.81, p < 0.0001). The effect of DM was highest at age 30−39 years (RR 3.05, 95% CI 2.35−3.96, p < 0.0001). In the group of <50 years old, DM patients had at least a twofold higher risk of developing uterine cancer (HR 2.39, 95% CI 2.09−2.74, p < 0.0001). Subjects among all ages diagnosed with polycystic ovary syndrome (PCOS) (HR 2.91, 95% CI 2.47−3.42, p < 0.0001), obesity (HR 2.13, 95% CI 1.88−2.41, p < 0.0001), and those undergoing hormone replacement therapy (HRT) (HR 1.60, 95% CI 1.33−1.93, p < 0.0001) were also positively associated with uterine cancer. Positive associations of hyperlipidemia (HR 1.33, 95% CI 1.22−1.46, p < 0.0001) and statin use (HR 1.27, 95% CI 1.12−1.44, p = 0.0002) on uterine cancer were only observed in subjects <50 years. On the contrary, hyperlipidemia was negatively associated with uterine cancer in subjects ≥50 years (HR 0.91, 95% CI: 0.84−0.98, p = 0.0122). Conclusions: DM is in general the most important risk factor for uterine cancer, especially in premenopausal women. Obesity, PCOS, HPL, statin use, and HRT were also associated with uterine cancer in subjects younger than 50 years. Premenopausal women with DM and respective comorbidities should be aware of the development of uterine cancer.

Mitochondrial Dysfunction Induced by High Estradiol Concentrations in Endometrial Epithelial Cells.

CONTEXT: A supraphysiological estradiol (E2) concentration after ovarian stimulation is known to result in lower embryo implantation rates in in vitro fertilization. Endometrial epithelial cell (EEC) apoptosis occurs after the stimulation with high E2 concentrations, and mitochondria play important roles in cell apoptosis. OBJECTIVE: To investigate the mitochondrial function in EECs after the stimulation with high E2 concentrations. MATERIALS AND METHODS: Human EECs were purified and cultured with different E2 concentrations (10-10, 10-9, 10-8, 10-7 M) in vitro, in which 10-7 M is supraphysiologically high. Eight-week-old female mouse endometrium was obtained 5.5 days after the injection of 1.25 IU or 20 IU equine chorionic gonadotropin, roughly during the embryo implantation window, to examine the in vivo effects of high E2 concentrations on mouse EECs. RESULTS: In vivo and in vitro experiments demonstrated decreased mitochondrial DNA contents and ATP formation after EECs were stimulated with supraphysiologically high E2 concentrations than those stimulated with a physiologic E2 concentration. Less prominent immunofluorescence mitochondrial staining, fewer mitochondria numbers under electron microscopy, lower 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide aggregate/monomer ratio, and greater reactive oxygen species (ROS) production were found after EECs were stimulated with supraphysiologically high E2 concentrations. The high E2-induced ROS production was reduced when EECs were pretreated with N-acetyl-cysteine in vitro, but remained unchanged after the pretreatment with coenzyme Q10. CONCLUSION: High E2 concentrations increase extramitochondrial ROS production in EECs and subsequently result in mitochondrial dysfunction.

Intentional endometrial injury increases embryo implantation potentials through enhanced endometrial angiogenesis†.

Embryo implantation rates have been found to be enhanced by precedent endometrial injuries, but the underlying mechanism is not fully investigated. Endometrial inflammation occurs both at peri-implantation period and after endometrial injury, in which vascular reaction is a distinctive feature of inflammation. In this study, intentional endometrial injury was done with a 0.7-mm-diameter brush inserted into the left uterine horn of female ICR mice, then turned around 720° (group 2), and the right uterine horn served as the controls without endometrial injuries (group 1). Intraperitoneal equine chorionic gonadotropin 2.5 IU was injected, followed by human chorionic gonadotropin 10 IU injection, and the uterus was dissected 5 days later, roughly at the peri-implantation period. The peri-implantation endometrium was obtained, and angiogenesis protein array revealed that matrix metalloproteinase-3 (MMP-3), plasminogen activator inhibitor-1 (PAI-1), insulin-like growth factor binding protein 1 (IGFBP-1), and IL-1α were more strongly expressed in injured endometrium (group 2) than in the controls (group 1). Immunohistochemical CD34 staining was more prominently expressed in group 2 uterus, and the treatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, significantly decreased CD34 immunopositive cells. The capabilities of permeability, proliferation, tube formation, and migration of mouse endometrial endothelial cells were significantly enhanced in group 2 than in group 1. Our results demonstrate that enhanced endometrial angiogenesis is a possible mechanism accounting for the increased endometrial receptivity after endometrial injury.

Iron suppresses ovarian granulosa cell proliferation and arrests cell cycle through regulating p38 mitogen-activated protein kinase/p53/p21 pathway.

Iron is an essential nutrient that may exert toxic effects when it accumulates in tissues. Little is known regarding its effects on gonadal function. Both Fe2+ and Fe3+ could be released from iron deposition. We employed mouse nonluteinized granulosa cell for in vitro studies and human ovarian tissues for Prussian blue and immunohistochemical staining to identify the iron deposition and effect in vivo. After treatment with FeSO4-7H2O or FeCl3 in granulosa cell cultured with follicle-stimulating hormone (FSH) for 48 h, we found that Fe2+ significantly suppressed FSH-induced granulosa cell proliferation and arrested the cell cycle at the G2/M phase by cell proliferation assay and flow cytometry. Fe2+ significantly increased intracellular reactive oxygen species (ROS) and ferritin levels of mouse granulosa cells. The increases in p21 and p53 messenger RNA and protein expression facilitated by Fe2+ treatment in mouse granulosa cells were significantly suppressed by separate treatments with p53 small interfering RNA and p38 mitogen-activated protein kinase (MAPK) inhibitors. An ROS inhibitor downregulated Fe2+-induced increases in p38MAPK expression in mouse granulosa cells. Quantitative analysis of immunohistochemical staining revealed that human ovarian tissue sections with positive Prussian blue staining had lower levels of proliferating cell nuclear antigen expression, but higher levels of p21, p53, and CDC25C expression than those with negative Prussian blue staining. Conclusively, Fe2+ could directly arrest the cell cycle and inhibit granulosa cell proliferation by regulating the ROS-mediated p38MAPK/p53/p21 pathway. Therefore, iron can directly affect female gonadal function.

High Estradiol Concentrations Induce Heat Shock Protein 70 Expression and Suppress Nuclear Factor Kappa B Activation in Human Endometrial Epithelial Cells.

The high serum estradiol (E2) concentrations induced during in vitro fertilization are detrimental to endometrial receptivity and may result in lower embryo implantation rates. We have previously found that high E2 concentrations inhibit the activation of nuclear factor kappa B (NF-kappa B), which led to endometrial epithelial cells (EECs) apoptosis. The objective of this study is to investigate the signaling pathways through which high E2 results in NF-kappa B downregulation in EECs. Isolated human EECs were cultured in different concentrations of E2 (10-10, 10-9, 10-8, 10-7 M). The expression of heat shock protein 70 (Hsp70) and heat shock factor 1 (HSF-1) were upregulated under supraphysiological E2 (10-7 M) concentration, whereas phosphorylated inhibitory kappa B-alpha (pI kappa B-alpha) and NF-kappa B p65 subunits were downregulated. Immunohistochemistry of C57BL/6 mouse EECs, that were exposed in vivo to high serum E2 from the administration of 20 IUs of equine chorionic gonadotropin, also demonstrated the same increase in HSF-1 and Hsp70 expression, and decrease in NF-kappa B. Immunoprecipitation of the induced Hsp70 proteins was achieved with the addition of inhibitory kappa B kinase gamma (IKK-gamma) antibodies, and elimination of this reaction occurred after addition of hsp70 siRNA. In conclusion, high E2 concentrations enhance HSF-1 and Hsp70 expression in EECs. The induced Hsp70 forms a complex with IKK-gamma and inhibits pI kappa B-alpha, which consequently suppresses NF-kappa B activation.

Frequency of low serum LH is associated with increased early pregnancy loss in IVF/ICSI cycles.

The role of LH during ovarian stimulation remains uncertain. Previous studies defined the low LH group using a single LH measurement on a predefined day of stimulation possibly not reflecting the entire follicular phase. This study retrospectively collected data from 619 IVF/ICSI cycles with GnRH antagonist and recombinant FSH. The low LH group was defined as LH concentration ≤0.8 mIU/ml at any time during the cycle. Pregnancy results were compared between patients with one episode of low LH or more than two episodes of low LH (study group) and those without low LH (control group). There was no difference in fertilization rates between the two groups (67.5 ± 1.7% versus 68.8 ± 1.0%, respectively). The implantation rates (20.4% versus 25.2%), clinical pregnancy rates (43.9% versus 45.2%) and live-birth rates (LBR) (23.7% versus 30.4%) appeared lower in the study group, but the differences were not significant. In the study group, there were significantly increased early pregnancy loss rates (31.1% versus 16.3%, P = 0.012). The odds of early pregnancy loss increases by 1.55 fold for increased episodes of low serum LH (P = 0.029). Whether the adverse outcome is due to impaired oocyte quality or an endometrial component deserves further investigation.

Factors Influencing the Recurrence Potential of Benign Endometrial Polyps after Hysteroscopic Polypectomy.

BACKGROUND: An endometrial polyp is a frequently encountered gynecologic disease with abnormal uterine bleeding and infertility being the two common presenting problems, and hysteroscopic polypectomy is an effective method to remove them. The postoperative polyp recurrence might result in reappearance of abnormal uterine bleeding or infertility, whereas factors influencing the postoperative recurrence potential have limited data. METHODS: This case-series report included 168 premenopausal women who suffered from endometrial polyps and underwent hysteroscopic polypectomy. All of them were awaiting a future pregnancy. Office hysteroscopy was done before and after hysteroscopic polypectomy, in which preoperative hysteroscopy examined the number, type, and location of endometrial polyps, and postoperative hysteroscopy checked the polyp recurrence. Surgical indications, either infertility or the presentation of abnormal uterine bleeding, and follow-up duration were recorded. RESULTS: Seventy-three out of 168 (43%) women had polyp recurrence after hysteroscopic polypectomy. Multivariate logistic regression analysis revealed that more endometrial polyps (P = 0.015) and longer duration of follow-up (P = 0.004) were significantly associated with an increased risk of postoperative polyp recurrence. The type of endometrial polyps was not correlated with polyp recurrence potential, whereas pedunculated type endometrial polyps were closely related to the presentation of abnormal uterine bleeding (P = 0.001). CONCLUSIONS: A higher number of endometrial polyps and longer follow-up duration are associated with a greater potential of polyp recurrence after hysteroscopic polypectomy.

Live birth after single embryo transfer of autologous cryopreserved oocytes from a patient with myelodysplastic syndrome who underwent allogenic peripheral blood stem cell transplantation.

We report a live birth after single embryo transfer derived from autologous cryopreserved oocytes of a patient with myelodysplastic syndrome who had undergone allogenic peripheral blood stem cell transplantation (PBSCT). In 2006, a 24-year-old female diagnosed with myelodysplastic syndrome was referred for fertility preservation before she underwent PBSCT. After controlled ovarian stimulation, 38 oocytes were retrieved for cryopreservation using a slow-freezing protocol. She was cured by PBSCT and entered menopause. After seven years, she requested thawing of the oocytes. She was prepared for a thawing cycle using hormone replacement therapy. Twenty-two cryopreserved oocytes were thawed, and 20 (91%) oocytes survived. Thirteen mature oocytes were inseminated by intracytoplasmic sperm injection. Ten (77%) oocytes were normally fertilized and 6 (60%) oocytes developed into blastocysts. Embryo transfer to her own uterus with one blastocyst was performed. Five blastocysts were vitrified. A sonographic exam at 7 weeks of gestation revealed one gestational sac with positive cardiac motion. A normal female baby weighing 2704 g was delivered at 40 weeks of gestation. A successful pregnancy from autologous cryopreserved oocytes is encouraging for cancer patients undergoing fertility preservation. For infertile cancer patients after PBSCT, we suggest the transfer of one embryo to reduce the risk of multiple pregnancies.

Current status of comprehensive chromosome screening for elective single-embryo transfer.

Most in vitro fertilization (IVF) experts and infertility patients agree that the most ideal assisted reproductive technology (ART) outcome is to have a healthy, full-term singleton born. To this end, the most reliable policy is the single-embryo transfer (SET). However, unsatisfactory results in IVF may result from plenty of factors, in which aneuploidy associated with advanced maternal age is a major hurdle. Throughout the past few years, we have got a big leap in advancement of the genetic screening of embryos on aneuploidy, translocation, or mutations. This facilitates a higher success rate in IVF accompanied by the policy of elective SET (eSET). As the cost is lowering while the scale of genome characterization continues to be up over the recent years, the contemporary technologies on trophectoderm biopsy and freezing-thaw, comprehensive chromosome screening (CCS) with eSET appear to be getting more and more popular for modern IVF centers. Furthermore, evidence has showen that, by these avant-garde techniques (trophectoderm biopsy, vitrification, and CCS), older infertile women with the help of eSET may have an opportunity to increase the success of their live birth rates approaching those reported in younger infertility patients.

Light-guided hysteroscopic resection of complete septate uterus with preservation of duplicated cervix.

The objective of the present study, performed at a tertiary university hospital, was to propose a novel method of hysteroscopic resection of complete septate uterus with preservation of duplicated cervix. The retrospective study included 5 women with complete septate uterus and cervical duplication and who also experienced infertility with or without pregnancy loss. All patients underwent bougie-guided or light-guided hysteroscopic perforation of the uterine septum above the endocervix, followed by septum resection. The success rate of complete uterine septum perforation under bougie guidance was 60% (3 of 5 procedures), and of light guidance was 100% (2 procedures). After hysteroscopic septum resection, 2 of 5 women achieved pregnancy within 3 months and delivered uneventfully at term. It is concluded that light guidance is superior to bougie guidance for hysteroscopic perforation of complete septate uterus with preservation of the duplicated cervix.

Blastocyst morphology score as an indicator of embryo competence for women aged younger than 38 years in in vitro fertilization cycles.

OBJECTIVE: This study aimed to clarify the usefulness of blastocyst scores and female age as embryo competence markers for embryo transfer in in vitro fertilization (IVF) cycles. MATERIALS AND METHODS: A total of 352 IVF cycles were investigated. The relevance of blastocyst scores and female age to pregnancy outcome was assessed by logistic regression analysis. RESULTS: We revealed that, for patients aged < 35 years, the score of the best embryo was the sole factor related to multiple pregnancy, whereas the score of the best two embryos was the only factor relevant to pregnancy. For patients aged 35-37 years, the score of the best three embryos was the sole factor correlated to both pregnancy and multiple pregnancy. As for older patients, the correlation between blastocyst morphology and pregnancy outcome was mainly affected by female age. CONCLUSION: The blastocyst score could be used to determine the number of blastocysts transferred to younger patients, but it is less useful for patients aged > 38 years. For older patients, female age is a better indicator to determine the number of transferred blastocysts.

Optimal waiting period for subsequent fertility treatment after various hysteroscopic surgeries.

OBJECTIVE: To investigate the endometrial wound healing duration after a hysteroscopic surgery. DESIGN: Prospective study. SETTING: Tertiary university hospital. PATIENT(S): One hundred sixty-three women who underwent hysteroscopic surgeries for endometrial polyp (n = 37), submucous myoma (n = 65), uterine septum (n = 16), and intrauterine adhesion (IUA; n = 45). INTERVENTION(S): Postoperative office hysteroscopy was consecutively done until complete endometrial wound healing. If there was newly formed IUA occurring at the endometrial wounds, adhesiolysis was immediately done with the tip of the office hysteroscope. MAIN OUTCOME MEASURE(S): Office hysteroscopic inspection of endometrial wound healing and the presence of newly formed IUA. RESULT(S): Thirty-two of 37 women (86%) achieved a fully healed endometrium 1 month after polypectomy, a higher rate than those after myomectomy (18%), septal incision (19%), and adhesiolysis (67%). Postoperative office hysteroscopy revealed that 88% and 76% of the women had new IUA formation after septal incision and adhesiolysis, respectively, more than those after myomectomy (40%) and polypectomy (0%). Women with postoperative new IUA formation were less likely to achieve endometrial wound healing within 1 month compared with those who had no new IUA formation (31% vs. 61%). CONCLUSION(S): The duration of endometrial wound healing is different after various hysteroscopic surgeries. Postoperative new IUA formation is an important factor influencing endometrial wound healing.

Prevention and management of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome is a relatively common complication of ovarian stimulation and can be life-threatening. The pathophysiology of ovarian hyperstimulation syndrome is characterised by increased capillary permeability, leading to leakage of fluid from the vascular compartment, with third space fluid accumulation and intravascular dehydration. The combined use of a gonadotrophin-releasing hormone antagonist protocol with gonadotrophin-releasing hormone agonist triggering and oocyte and embryo freezing has considerable promise in preventing ovarian hyperstimulation syndrome. Women with severe ovarian hyperstimulation syndrome require hospitalisation for more careful monitoring and treatment. Transvaginal paracentesis can be used as an outpatient treatment to prevent the need for hospitalisation. The inhibition of vascular permeability seems to be a novel therapeutic approach to preventing and treating ovarian hyperstimulation syndrome.

Update on management of ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is a relatively common complication of ovarian stimulation and can be life threatening. The pathophysiology of OHSS is characterized by increased capillary permeability, leading to leakage of fluid from the vascular compartment, with third-space fluid accumulation and intravascular dehydration. The increased intra-abdominal pressure indicated that OHSS may be considered a compartment syndrome. Vascular endothelial growth factor, also known as vascular permeability factor, has emerged as one of the mediators intrinsic to the development of OHSS. Conventional management is focused on supportive care until the spontaneous resolution of the condition. The standard of care for treatment-monitoring of appropriate clinical parameters, fluid balance management, thrombosis prophylaxis, and ascites treatment-should prevent severe morbidity in most cases. This review will cover inpatient and outpatient management. The potential therapeutic approach targeting the vascular endothelial growth factor system will be discussed.

Impact of submucous myoma on the severity of anemia.

OBJECTIVE: To analyze the impact of the diameter (myoma-φ) and the uterine cavity protruding proportion (protruding %) of the submucous myoma on serum hemoglobin (Hb) level. DESIGN: Retrospective descriptive study. SETTING: Tertiary university hospital. PATIENT(S): Two hundred fifty-nine women with a single submucous myoma. INTERVENTION(S): Transvaginal ultrasound examination and office hysteroscopy. MAIN OUTCOME MEASURE(S): Myoma-φ and protruding % were determined with transvaginal ultrasound scan and office hysteroscopy. Menorrhagic period (months) and serum Hb were recorded within 2 weeks of these examinations. RESULT(S): Serum Hb negatively correlated with myoma-φ, protruding %, and menorrhagic period. The negative association between serum Hb and protruding % was even more prominent after adjusting for myoma-φ. Multivariate regression analysis revealed that myoma-φ and protruding %, after adjusting for the confounding effect of menorrhagic period, maintained a strongly negative correlation with serum Hb level. When myoma-φ was <2 cm, however, the serum Hb levels were similar regardless of the protruding %. CONCLUSION(S): For women with a single submucous myoma, myoma-φ and protruding % can determine the serum Hb level significantly . As myoma-φ and protruding % increased, the serum Hb level decreased accordingly.

High serum dehydroepiandrosterone sulfate is associated with phenotypic acne and a reduced risk of abdominal obesity in women with polycystic ovary syndrome.

BACKGROUND: Women with polycystic ovary syndrome (PCOS) are known to have high prevalence of acne and elevated androgen levels. The current study aims to determine if dehydroepiandrosterone sulfate (DHEAS) level is associated with the presence of acne and reduced risk of abdominal obesity in women with PCOS, after considering the concurrent high testosterone level and insulin resistance (IR). METHODS: Three hundred and eighteen untreated consecutive Taiwanese women with PCOS were enrolled. Phenotypic hyperandrogenism was recorded, and BMI, waist circumference, waist-to-hip ratio, lipid profiles, fasting glucose and insulin levels and hormone profiles were measured. RESULTS: Women with acne were younger, had higher serum DHEAS levels (6.01 ± 3.45 versus 4.87 ± 2.49 µmol/l, P = 0.002) and a lower BMI (P = 0.0006), but comparable serum testosterone levels, in comparison with women without acne. The aggravating effect of elevated DHEAS on the risk of acne (odds ratio = 2.15, 95% confidence interval: 1.25-3.68, P = 0.005 for DHEAS cut-off of 6.68 µmol/l) still exited after adjustment for age and BMI. The DHEAS level was positively correlated with the testosterone level, but inversely related to waist circumference, waist-to-hip ratio, BMI, IR index, low-density lipoprotein-cholesterol and triglycerides. Women with PCOS in the highest quartile of DHEAS had the lowest risk of abdominal obesity after adjustment for age, IR, dyslipidemia, testosterone and estradiol levels. CONCLUSIONS: Our results demonstrated the high serum DHEAS in women with PCOS was associated with the presence of acne and a significantly reduced risk of abdominal obesity, independent of serum testosterone concentration and IR.

Signal mechanisms of vascular endothelial growth factor and interleukin-8 in ovarian hyperstimulation syndrome: dopamine targets their common pathways.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a serious complication of ovarian stimulation with massive ascites, pleural effusion and hemoconcentration. The pathophysiological signal mechanisms of OHSS are still unclear and merit further investigation. METHODS: Various angiogenic cytokines of follicular fluid and ascites of patients with risk of OHSS were measured, and examined for inducing endothelial permeability. These include vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, basic fibroblast growth factor, tumor necrosis factor-alpha, IL-1alpha, IL-1beta and platelet-derived growth factor. We explore the molecular signal pathways of major contributing cytokines in granulosa-lutein cells and endothelial cells possibly involved in OHSS. RESULTS: Neutralizing antibodies of VEGF or IL-8 significantly decreased follicular fluid- and ascites-induced endothelial permeability. Human chorionic gonadotrophin induced VEGF secretion of granulosa-lutein cells through the Sp1 and CREB dependent pathways. IL-8 activated CXCR1/2 of endothelial cells leading to VEGF receptor (VEGFR)-2 transactivation. Both VEGF and IL-8 of follicular fluid enhanced endothelial permeability via VEGFR-2-mediated Rho/Rock activation, actin polymerization and phosphorylations of VE-cadherin and occludin, resulting in opening of adherens junctions and tight junctions. Dopamine (2 microM) inhibited follicular fluid-induced VEGFR-2 signals and endothelial permeability, without diminishing migration and tube formation. CONCLUSIONS: Our results suggest that VEGF and IL-8 secreted from corpora luteae may play major roles in OHSS. Delineation of signal pathways would be helpful for treatment. Dopamine may block VEGF- and IL-8-induced endothelial permeability by inhibiting common VEGFR-2 dependent signals.

Delineation of an isodicentric Y chromosome in a mosaic 45,X/46,X,idic(Y)(qter-p11.3::p11.3-qter) fetus by SRY sequencing, G-banding, FISH, SKY and study of distribution in different tissues.

Many factors such as genetic, developmental and hormonal are involved in mammalian sex determination. The relative importance and the mutual interactions among those factors are obscure. Study of cytogenetic mosaicism involving sex chromosomes may help to further unravel the mysterious process. We report a fetus with a mosaic karyotype, 45,X/46,X,idic(Y)(qter-p11.3::p11.3-qter), with unambiguous male external genitalia and a defect in the interventricular septum of the heart. Genotype of this fetus was extensively studied by technologies including sequencing of SRY (sex-determining region on the Y chromosome) gene, G-banding, FISH (fluorescence in situ hybridization) and SKY (spectral karyotyping). A markedly higher percentage of Y-containing cells was observed in the gonads (55%) than in the amniotic fluid (17%) and placental villi (11%), which was considered to be the major reason why the fetus did not have ambiguous genitalia.