Genetics and human conception: DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease

We report the world's first clinical pregnancy resulting fromDNA-based enrichment for X-bearing human spermatozoa, for preventionof X-linked hydrocephalus. Sperm separation was followed byembryo biopsy and nested multiplex polymerase chain reaction(PCR) for gender determination. Enriched populations of X-bearingspermatozoa ranging from 80 to 89% pure as determined by fluorescencein-situ hybridization (FISH) resulted in in-vitro fertilization(IVF) rates indistinguishable from normal IVF procedures (65%).In two separate biopsy procedures, 7/9 and 15/16 of the resultingembryos were determined to be female by multiplex PCR. Embryotransfer resulted in a karyotypically normal female fetus. Thistechnique should be widely applicable to gender selection forthe prevention of genetic disorders.     

Development of the fetal uterus between 19 and 38 weeks of gestation: in-utero ultrasonographic measurements.

In-utero assessment of the internal female genitalia is important for determination of fetal gender in fetuses with suspected genital tract anomalies. We therefore measured fetal uterine transverse width and circumference from 19 weeks of gestation until term, using transvaginal and transabdominal high-resolution ultrasound techniques in order to establish nomograms. A prospective, cross-sectional study on 180 normal singleton pregnancies was performed. Data were obtained for 140 normal fetuses. The mean +/- SD uterine width and circumference were 12.9 +/- 4.1 mm (95% confidence interval 12.1-13.7), and 40.2 +/- 12.5 mm (95% confidence interval 37.9-42.5) respectively. Uterine size as a function of gestational age was expressed by the regression equations: uterine width (mm) = 12.9 + 0.7 x gestational age (weeks), and uterine circumference (mm) = 40.2 + 2.1 x gestational age. The correlation coefficients, r = 0.885 and r = 0.888, for uterine width and circumference, by gestational age respectively, were highly statistically significant (P < 0.001). A nomogram of uterine width and circumference per gestational week, and the 95% prediction limits were defined. The present data offer baseline measurements of the fetal uterus that may allow intrauterine assessment of the female genital tract and associated fetal gender.     

Correlation of maternal plasma total cell-free DNA and fetal DNA levels with short term outcome of first-trimester vaginal bleeding

BACKGROUND: The current methods using sonographic parameters and/or maternal serum beta-HCG levels to predict spontaneous abortion are not satisfactory. The aim of this study was to determine whether maternal plasma fetal DNA and total DNA levels could be used to predict spontaneous abortion. METHODS: We prospectively studied pregnant women who presented with vaginal bleeding in the first trimester of pregnancy, and those who had no vaginal bleeding (controls). DYS14 and the beta-globin gene were used to measure the maternal plasma levels of fetal and total DNA, respectively, by real-time PCR. RESULTS: A total of 1114 women were studied. Both maternal plasma fetal and total DNA concentrations increased with gestation from 6 to 11.6 weeks in the controls. The multiple of medians (MoMs) of fetal and total DNA concentration in those who miscarried were significantly greater (P < 0.001) than in the normal controls by about 5- and 4-fold respectively. Using a cut-off value of 1.6 MoMs for total DNA to predict spontaneous abortion, the sensitivity was 98.2% and false positive rate was 4.7%. However, using a cut-off value of 1.8 MoMs for fetal DNA, the corresponding figures were 97% and 44.3%, respectively. CONCLUSIONS: Both maternal plasma fetal and total DNA concentrations increased throughout the first trimester. Significantly high levels of fetal and total DNA were found in those who miscarried.     

抑制素A水平与中孕期筛查标志物及影响因素的相关性分析

目的评估中孕期母亲血清中抑制素A水平与其它血清标志物和影响因素的相关性。方法选择2008年3月-2010年12月在广州市妇女儿童医疗中心接受中孕期＂三联＂唐氏筛查的单胎妊娠孕妇共2802例。采用全自动酶联免疫化学发光分析法检测血清中抑制素A水平。结果 （1）在正常妊娠孕妇血清样本,抑制素A与中孕期血清标志物freeβ-hCG、AFP相关且有统计学意义（P〈0.01）,尤其与freeβ-hCG的相关性最高（r=0.502）,而与uE3相关性没有统计学意义（P〉0.05）;在胎儿唐氏综合征孕妇血清样本,抑制素A与freeβ-hCG、AFP和uE3的相关性均没有统计学意义（P〉0.05）。（2）135例阴道流血和40例糖尿病孕妇血清中抑制素A水平与无合并症的孕妇没有差异（P均〉0.05）。孕期母亲体重与血清中抑制素A水平呈负相关（r=-0.101）,并有统计学意义（P〈0.01）。男性胎儿孕妇血清中抑制素A水平低于女性胎儿孕妇并有统计学意义（P〈0.01）。结论血清中抑制素A水平与中孕期标志物freeβ-hCG的相关性最高,与母亲体重呈负相关性,且与胎儿性别相关,但不受孕期母亲阴道流血或糖尿病的影响。     

Sidestream smoking is equally as damaging as mainstream smoking on IVF outcomes

BACKGROUND: Cigarette smoking (CS) is a widely recognized health hazard, yet it remains prevalent in society and the effects of environmental tobacco smoke exposure on fertility are unknown. Our objective was to measure the effects of CS on the fertility of mainstream (MS) or sidestream (SS) smoke-exposed women compared to their non-smoking (NS) counterparts. METHODS: This retrospective study investigated 225 female patients undergoing IVF (n = 97) or ICSI (n = 128). Patients were grouped based on their smoking status for comparison. This included: 39 MS (18 IVF and 21 ICSI); 40 SS (16 IVF and 24 ICSI); and 146 NS (63 IVF and 83 ICSI) women. Fertility treatment outcomes including embryo quality, implantation and pregnancy rate were measured. RESULTS: No difference in embryo quality between the three groups was observed. However, there was a significant difference in implantation rate (MS = 12.0%, SS = 12.6%, and NS = 25.0%) and pregnancy rate (MS = 19.4%, SS = 20.0%, and NS = 48.3%) per embryo transfer. CONCLUSIONS: Despite similar embryo quality there was a striking difference in implantation and pregnancy rates of MS and SS smokers when compared with NS. Our data demonstrate that the effects of SS smoking are equally as damaging as MS smoke on fertility.     

Delivery of a severely anaemic fetus after partial molar pregnancy: clinical and ultrasonographic findings

The incidence of a normal live fetus and a partial molar placenta is extremely rare. Although triploidy is the most frequent association, a fetus with normal karyotype can survive in cases of partial molar pregnancy. We report a case of partial molar placenta in which a live female baby was delivered at 32 weeks gestation by a 30-year-old woman. At the 18th week, ultrasonographic examination revealed a normal fetus with a huge, multicystic placenta. Chromosomal evaluation by amniocentesis revealed a normal female karyotype (46,XX), and serial biometric measurement of the fetus showed normal growth during pregnancy. There were no obstetric complications until the 32nd gestational week when preterm rupture of the membranes occurred. The electronic fetal heart beat tracing showed a repeated sinusoid pattern and late deceleration after admission. The patient underwent emergency Caesarean section and delivered a 1551-g, anaemic female baby with an Apgar score of 1, 4 and 6 at 1, 5 and 10 min, respectively. The baby recovered within 2 weeks after respiratory support and transfusion of packed red blood cells. Although anaemia is one of the risk factors that jeopardize the fetus in the case of partial molar pregnancy, termination is not indicated when the fetus is normal and no complications have occurred.     

用荧光原位杂交从母血中检测胎儿细胞

目的 从母血中分离胎儿细胞并确定其来自胎儿。方法 从孕早、中期各２０名、分娩后１５名母血中富集并分离有核细胞。用Ｙ特异性探针（ＰＹ３．４）行荧光原位杂交,从中识别胎儿细胞。结果 孕早、中期孕妇各怀１５名男胎。阳性细胞比例是１：６５２８．０及１：２７３．８。与同期１０名女胎阳性细胞相比,差别有高度显著性。分娩１周内的３名,阳必民孕中期的差别没有显著性。分娩３个月后的阳性率与比,差别有高度显著性。分娩     

Ontogeny of the Fetal Immune System: Study on Pregnancies with Rh-Isoimmunization and Nonimmune Fetal Hydrops

This study aims at observing and comparing the antigen expression of some fetal T- and B-lymphocyte subpopulations in Rh-isoimmunization, which determines anemic hypoxia in the fetus, and nonimmune fetal hydrops (NIFH) which, even if there are some etiological factors involved, causes hipoxic hypoxia in the fetus. Twelve fetuses were studied by way of 30 fetal blood samples obtained by ultrasound-guided cordocentesis between the 20th and 36th gestational week. Twenty-four blood samples in all where taken from the eight fetuses with Rh-isoimmunization. Six blood samples were obtained from the four fetuses with NIFH. The lymphocyte phenotypes studied by monoclonal antibodies and flow cytometry were the following: CD3, CD4, CD8, expression of T-lymphocyte subpopulations; BsIg, CD19, expression of B-lymphocyte subpopulations. We observed a near-normal maturation process in fetuses with Rh isoimmunization, whereas in fetuses with NIFH we observed inhibition and/or delayed expression of T-lymphocytes. An early and increased B-lymphocyte activation marked a cooperation between the two systems in the early gestational periods.     

Noninvasive prenatal screening for RHD: the Stockholm study

The non-invasive technique to determine fetal RHD status opens the opportunity to change the antenatal screening and Rh-prophylaxis programs. During the period September 2009 to December 2011, we performed a study in the Stockholm area with approximately 27000 pregnancies per year. The study included routine cell free fetal DNA (cffDNA) RHD genotyping in early pregnancy followed by targeted RAADP in gestational week 29 to all RhD negative pregnant women carrying an RHD positive fetus. The new approach in our strategy, compared to previous studies, was that fetal RHD screening was done in early pregnancy at the first antenatal visit and based on a single-exon 4 assay. The implementation of this new screening program in a routine clinical setting is described. The final results of the study are still under analysis. The conclusion until now is that fetal RHD screening in early pregnancy is feasible and accurate with a high sensitivity and specificity, provided samples before gestational week eight were excluded.     

A prospective study of the effects of female and male caffeine consumption on the reproductive endpoints of IVF and gamete intra-Fallopian transfer

BACKGROUND: This study evaluated the timing and amount of caffeine intake by women and men undergoing IVF and gamete intra-Fallopian transfer (GIFT) on oocyte retrieval, sperm parameters, fertilization, multiple gestations, miscarriage, and live births. METHODS: A prospective study of 221 couples was conducted in Southern California between 1993 and 1998. "Usual" caffeine intake during lifetime and 1 year prior to attempt, caffeine intake during the week of the initial clinic visit, as well as intake during the week of the procedure, was evaluated from beverages (coffee, soda, tea) and chocolates. RESULTS: Not achieving a live birth was significantly associated with "usual" female caffeine consumption [adjusted odds ratios (95% confidence intervals): 3.1 (1.1, 9.7) and 3.9 (1.3, 11.6) for intake of >2-50 and 50 mg/day, compared with 0-2 mg/day] and consumption during the week of the initial visit [2.9 (1.1, 7.5) and 3.8 (1.4, 10.7)] female compared with 0-2 mg/day, although caffeine use was low. Infant gestational age decreased by 3.8 (-6.9, -0.7) or 3.5 (-6.7, -0.3) weeks for women who consumed >50 mg/day of caffeine "usually" or during the week of the initial visit. The odds of having multiple gestations increased by 2.2 (1.1, 4.4) and 3.0 (1.2, 7.4) for men who increased their "usual" intake or intake during the week of the initial visit by an extra 100 mg/day. Caffeine intake was not significantly associated with other outcomes. CONCLUSIONS: This is the first IVF/GIFT study to report any effect of caffeine on live births, gestational age, and multiple gestations. If these findings are replicated, caffeine use should be minimized prior to and while undergoing IVF/GIFT.     

Placental Transport of Maternal Immunoglobulin G

PROBLEM: How does the placental transport of immunoglobulin G (IgG) vary with gestational age? METHOD OF STUDY: MEDLINE was searched for the appearance of “pregnancy” and “IgG” in the title or abstract from 1966 up to 1995. All publications were reviewed for any kind of IgG data from the fetus and the mother and “known” gestational age. In total, 96 publications from the MEDLINE search fulfilled the search criteria. Seven of ninety-six publications contained IgG and gestational age data from the fetus and/or the mother. Five other publications that fulfilled the same criteria were also available and were also included. RESULTS: Fetal serum IgG concentrations were found to increase with gestational age according to the formula: fetal IgG = 13.564 ? 1.094* gestational age (GA) + 0.026* GA² (R² = 0.877). The fetomaternal (f/m) ratio was also found to increase with gestational age according to the formula: f/m IgG = 0.399 ? 0.059* GA + 0.003* GA² ? 2.065* 10^—5* GA³ (R² = 0.831). CONCLUSIONS: The fetal IgG concentrations exceed maternal IgG concentrations after the 35th week of pregnancy. The fetal IgG concentrations are extremely low in the beginning of pregnancy, they exceed 2.5 g/L at >25 pregnancy weeks, and they reach 5 g/L at pregnancy week 30.     

Vanishing twins: a predictor of small-for-gestational age in IVF singletons

BACKGROUND: The purpose of this study was to assess the effect of a vanishing twin on the risk of being small-for-gestational age (SGA) in in vitro fertilization (IVF) singletons. METHODS: The study included 642 survivors of a vanished co-twin, 5237 primary singletons and 3678 primary twins. The survivor cohort was subdivided according to gestational age at the time of vanishing to give groups of early (<8 weeks), intermediate (8-22 weeks) and late (>22 weeks) survivors. RESULTS: The rate of SGA infants was significantly higher in survivors than in singletons (OR: 1.50, 95%CI: 1.03-2.20) and a significant inverse correlation was observed between SGA and the gestational age at the time of vanishing (r = -0.10, P < 0.02). Also in term infants, the risk of birthweight <2500 g was higher in survivors than in singletons (OR: 1.71, 95%CI: 1.06-2.74). A similar increase in the rate of low birthweight in term survivors was seen with increasing gestational age at the time of vanishing (r = -0.12; P < 0.01). In multiple logistic regression analysis adjusting for maternal age, parity, child gender and pre-eclampsia, the vanishing of a co-twin (OR: 1.56, 95%CI: 1.06-2.27) and gestational age at the time of vanishing (OR: 2.08, 95%CI: 1.00-4.35) were the only significant predictors of being SGA. CONCLUSIONS: IVF singletons with a vanished co-twin had a higher rate of SGA than singletons from a single gestation and the risk of SGA is increased with increasing gestational age at the time of vanishing.     

DNA-based X-enriched sperm separation as an adjunct to preimplantation genetic testing for the prevention of X-linked disease

We report the world's first clinical pregnancy resulting fromDNA-based enrichment for X-bearing human spermatozoa, for preventionof X-linked hydrocephalus. Sperm separation was followed byembryo biopsy and nested multiplex polymerase chain reaction(PCR) for gender determination. Enriched populations of X-bearingspermatozoa ranging from 80 to 89% pure as determined by fluorescencein-srtu hybridization (FISH) resulted in in-vitro fertilization(IVF) rates indistinguishable from normal IVF procedures (65%).In two separate biopsy procedures, 7/9 and 15/16 of the resultingembryos were determined to be female by multiplex PCR. Embryotransfer resulted in a karyotypically normal female fetus. Thistechnique should be widely applicable to gender selection forthe prevention of genetic disorders. flow cytometry/fluorescence in-situ hybridization/gender selection/hereditary diseases/spermatozoa     

自然流产组织应用FISH技术的染色体异常遗传因素分析

目的分析兰州地区自然流产组织中的染色体异常发生率,并探讨染色体异常与自然流产的年龄、孕周、次数、胚胎性别的关系。方法应用荧光原位杂交（fluorescent in situ hybridization,FISH）技术检测了299例自然流产的绒毛或胚胎组织。结果我们检测出了119例染色体异常病例,其中16-三体最为常见,占所有异常总数的27．73％,X单体次之,占18．49％。我们还发现自然流产与流产时的妊娠孕周和胚胎性别有关。结论染色体异常是自然流产的一个重要原因,尽早地遗传咨询能够有效的降低出生缺陷的发生率。     

Fetal gender impact on multiple-marker screening results

Maternal serum alpha-fetoprotein (MSAFP), human chorionic gonadotropin (hCG), and unconjugated estriol (uE3) are used in combination with maternal age to calculate the risk for Down syndrome (DS) in pregnancy. Increased levels of hCG and decreased levels of MSAFP and uE3 are consistent with an increased risk for DS. We retrospectively evaluated second-trimester maternal serum marker levels in a large cohort of patients with known normal outcomes and documented fetal gender. These included 15,428 patients who had MSAFP measurements, 11,428 patients with both MSAFP and hCG, and 6,090 patients with all three markers including uE3. MSAFP levels in patients with female fetuses were consistently lower than those with males. Conversely, hCG was higher in pregnancies with females as compared to males. No gender-related difference was noted for uE3. These results would suggest that the computed DS risk for female fetuses is higher than for males, despite the fact that the incidence of DS is similar in both genders. This information could be useful for calculating gender-specific DS risk; however, this would require ultrasonographic determination of fetal sex. Am. J. Med. Genet. 76:369–371, 1998. © 1998 Wiley-Liss, Inc.     

环孢霉素A对小鼠妊娠失败模型妊娠预后的影响

目的 :探讨环孢霉素A(CyclosporinA ,CsA)对小鼠妊娠失败模型及正常妊娠模型妊娠预后的影响。方法 :于孕 4d分别给小鼠妊娠失败模型及正常妊娠模型腹腔注射不同剂量 (0 ,5 ,10 ,15mg kg)CsA ,于孕 14d处死各组小鼠 ,观察各实验组胚胎吸收率、胎鼠及胎盘重量变化。结果 :①CsA可使小鼠妊娠失败模型胚胎吸收率显著降低 ,接近于小鼠正常模型胚胎吸收率 ;同时胎鼠及胎盘重量无显著性差异 ,但有增加趋势。②CsA对小鼠正常妊娠模型胚胎吸收率无明显影响 ;胎鼠及胎盘重量亦无显著性差异。结论 :于孕早期 (胚胎着床期 )腹腔注射CsA可使小鼠妊娠失败模型胚胎吸收率恢复至正常水平 ,从而可能成为保胎的潜在药物。     

A 47,XXY fetus conceived after ICSI of spermatozoa from a patient with non-mosaic Klinefelter's syndrome: case report

The birth of 12 healthy infants to fathers with non-mosaic Klinefelter's syndrome has been reported so far. The spermatozoa for these pregnancies was obtained from frozen-thawed ejaculate in one pregnancy (twins) and from the testis in the remaining 10 infants. All of them had a normal karyotype. We describe a patient with non-mosaic Klinefelter's syndrome from whom a testicular biopsy was obtained and motile spermatozoa were collected. Of 16 oocytes that were injected, 14 fertilized and cleaved. Three embryos were transferred, resulting in a triplet pregnancy. Karyotype analysis from chorionic villous sampling revealed 46,XX, 46,XY and 46,XXY from the three fetuses. The affected 46,XXY fetus was reduced on the 14th gestational week. The pregnancy culminated with the birth of a healthy male and female, on the 36th gestational week, weighing 3600 and 2660 g respectively. This case report proves the presence of hyperploid spermatozoa in the seminiferous lumen, and strengthens the necessity of genetic diagnosis of the embryos or fetuses in such pregnancies to fathers with non-mosaic Klinefelter's syndrome.     

Blood lymphocyte chimerism associated with IVF and monochorionic dizygous twinning: case report

We report on dizygotic (DZ) twins, conceived by IVF and ICSI with assisted hatching, who each had a mixture of 46,XX and 46,XY cells in blood lymphocytes. The female twin had mild genitalia abnormalities but further study revealed anatomically normal reproductive anatomy. Chromosome and fluorescence in situ hybridization studies of buccal, skin and ovarian tissue were normal, as were buccal tissue DNA studies. Fetal ultrasound and fetal membrane pathology were consistent with a monochorionic, diamniotic placenta (MCDAP). These twins thus have blood chimerism but are not chimeric in the other tissues studied. The mechanism for the chimerism could be due to either placental vascular anastamoses (after the development of the haematoblast stem cells) or due to an admixture of trophoblast cells during early blastocyst development. Such trophoblast cell admixtures would be restricted to the extraembryonic tissues so that general physical development in the fetus is normal and without somatic cell chimerism. This case in combination with others previously reported suggests that in IVF conceptions, the prevalence of blood chimerism associated with twinning, and the occurrence of DZ twinning associated with MCDAP, may be higher than previously thought.     

Clinical applications of fetal sex determination in maternal blood in a preimplantation genetic diagnosis centre

BACKGROUND: Couples with a risk of transmitting X-linked diseases who are included in a preimplantation genetic diagnosis (PGD) programme need early and rapid fetal sex determination in two situations. The first situation is for the control of embryo sexing after PGD and the second situation is for those couples having a spontaneous pregnancy before the start of their PGD cycle. Among invasive techniques, chorionic villus sampling is the earliest procedure for fetal sex determination and molecular analysis of X-linked genetic disorders during the first trimester but it is associated with a risk of fetal loss. Non-invasive procedures such as ultrasound examination allow reliable fetal sex determination only during the second trimester. Reliable fetal sex determination can now be realised by using SRY gene amplification in maternal blood. METHODS AND RESULTS: We report the use of fetal sex determination from maternal serum as a diagnostic tool for the control of embryo sexing (two cases) and to manage spontaneous pregnancies in couples included in a PGD programme for X-linked diseases (five cases). Fetal sex determination using SRY gene amplification in maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis or ultrasound examination and at birth. This novel strategy allowed the PGD results to be controlled precociously and avoided the performance of invasive procedures in four cases of female fetus. CONCLUSIONS: This rapid fetal sex determination during the first trimester provides advantages to both clinicians and patients in a PGD centre.     

孕中晚期胎儿小脑生长发育规律及其与单纯后颅窝池增宽关系MRI研究

目的　利用MRI测量孕中晚期正常胎儿小脑横径及体积,研究其随孕周的发育规律,并探讨其在单纯后颅窝池增宽诊断中的临床价值。 方法　本研究前瞻性纳入139例孕周为24~38周的正常胎儿,行胎儿颅脑1.5T MRI检查,在三维后处理工作站（OsiriX软件）上测量小脑横径（TCD）、体积（CV）,分析其随孕周变化的规律;另外同期纳入45例经产前超声及磁共振诊断为胎儿单纯后颅窝池增宽作为研究组,将其按孕周划分为两组（I组：28~32周,20例;II组,33~38周,25例）,同时按孕周及例数匹配45例正常胎儿作为对照组,定量分析单纯后颅窝池增宽时胎儿小脑发育的情况。 结果　①胎儿小脑横径与孕周呈线性增长,直线方程为TCD=-1.746+0.182GA（R2=0.953,P<0.05）,胎儿小脑体积与孕周呈指数增长,曲线方程为CV=0.072exp（0.151GA）（R2=0.957,P<0.05）;②单纯后颅窝池增宽胎儿与正常胎儿相比分析得出：I组,研究组胎儿小脑横径及体积与对照组间对比无统计学差异（P均>0.05）;II组,研究组胎儿小脑横径及体积与对照组对比无统计学差异（P均>0.05）。 结论　孕中晚期正常胎儿小脑横径及体积随孕周增加分别呈线性及指数增长趋势,此结果有助于评估胎儿小脑发育程度;胎儿单纯后颅窝池增宽与小脑横径及体积无明显相关性,这有助于对后颅窝池增宽的临床意义再认识。