复发性流产和不明原因不孕患者子宫内膜白血病抑制因子表达研究

目的探讨白血病抑制因子(leukaemia inhibitory factor,LIF)在复发性流产和不明原因不孕患者黄体中期子宫内膜的表达情况.方法采用实时荧光定量PCR技术,对31例不明原因不孕和30例复发性流产患者黄体中期子宫内膜LIF表达量的测定,对照组34例为继发性不孕患者(输卵管阻塞的)黄体中期子宫内膜.结果不明原因不孕组妇女子宫内膜中LIF表达量(M)为(169.00ng/l),与对照组(M) 为(240.00ng/l)相比,差异有显著性(P<0.01);习惯性流产组LIF表达量(M) 为(90.25ng/l),与对照组相比,差异有显著性(P<0.01),复发性流产组与不明原因不孕组相比差异无显著性(P>0.05).结论 LIF基因可能在启动胚泡着床和维持妊娠方面有重要作用, 黄体中期子宫内膜LIF基因表达量的减少可能是导致不明原因不孕和复发性流产的原因.     

Molecular genetic studies of HLA-DRB1 alleles in patients with unexplained recurrent abortion in the Japanese population

BACKGROUND: Recently, evidence that HLA antigens are markers for recurrent spontaneous abortion has gained increased attention. Although the association between HLA class II antigens and patients with unexplained recurrent abortion was elucidated by a large population study in a Caucasian population, such analyses have been conducted in only a small Japanese population. The aim of the present study was to determine whether HLA-DR antigens are associated with patient populations with unexplained recurrent abortion in the Japanese population. METHODS: HLA-DRB1 genotypes were determined using a PCR-restriction fragment length polymorphism (PCR-RFLP) method in 93 patients with unexplained recurrent abortion (79 primary recurrent aborters and 14 secondary recurrent aborters) and in 115 normal fertile women. The rate of possession of each HLA-DRB1 genotype was compared among the three populations. RESULTS: The rate of possession of the HLA-DRB1*1502 in patients with secondary recurrent abortion was significantly higher (P < 0.01 after correction for multiple comparisons) compared with the control, fertile women. The rate of possession of HLA-DRB1*1502 was also higher in patients with primary recurrent abortions than in controls, but the difference was not statistically significant after correction. CONCLUSIONS: These findings suggest that HLA-DRB1*1502 might be a risk allele for unexplained recurrent abortion in the Japanese population.     

Human chorionic gonadortrophin measurements in the luteal phase of the menstrual cycle of infertile and fertile women

Serum human chorionic gonadotrophin (HCG) determinations wereperformed every other day during the luteal phase of the menstrualcycle of 20 women suffering from unexplained infertility andof 20 women assumed to be fertile. HCG was determined by a specificradioimmunoassay which applied an antiserum directed againstthe HCG subunit. The group of infertile patients demonstrateddetectable serum HCG in only three cases. No clinically confirmedpregnancies were achieved in this group. Eighteen of the 20women presumed to be fertile showed detectable HCG levels. Fiveof these achieved ongoing pregnancies, while 13 women revealedincreased HCG levels without delayed menstrual periods. Thefirst day of detection of HCG during the luteal phase did notdiffer between the two groups. In the five women with ongoingpregnancies the time of detection of HCG ranged from day 5 untilday 12 after ovulation. It is concluded that the high rate ofincreased HCG values in the fertile group may represent earlyconceptual loss, but the apparently disorderly HCG results indicatethe need for cautious interpretation of HCG determinations duringthe luteal phase. Furthermore, early conceptual loss forms nomajor explanation for unexplained infertility.     

不孕及反复自然流产患者血清抗精子抗体分析

目的：探讨不孕及反复自然流产与抗精子抗体（AsAb）的关系。方法：用金标法检测有反复自然流产史患者245例（流产组）、原发或继发不孕患者164例（不孕组）及正常妇女40例（对照组）血清中的AsAb。结果：不孕组AsAb总阳性率52.44%,流产组总阳性率48.57%,与对照组比较差异均有统计学意义（P〈0.05）。结论：AsAb与不孕及反复自然流产有着密切关系。     

抗子宫内膜抗体和抗精子抗体在临床中的价值探讨

目的探讨抗精子抗体和抗子宫内膜抗体在临床上检测原发性不孕和复发性自然流产的价值。方法回顾性分析2018年1月至2018年12月在复旦大学附属妇产科医院诊治的原发性不孕患者及复发性自然流产患者病史,以同期进行常规检查的孕妇作为对照,对比研究对象抗子宫内膜抗体、抗精子抗体的水平,分析抗子宫内膜抗体、抗精子抗体在原发性不孕和复发性自然流产中可能的相关性。结果收集到原发性不孕患者170例及复发性自然流产患者238例病史资料,以同期进行常规检查的孕妇208例作为对照组。原发性不孕患者抗精子抗体检测阳性例数显著高于对照组,但抗子宫内膜抗体并无显著改变;复发性自然流产患者抗子宫内膜抗体检测阳性率显著高于对照组,但抗精子抗体并无明显不同;早孕期流产患者抗精子抗体检测阳性率显著高于对照组;发生复发性自然流产在4次以上的病人,其体内抗子宫内膜抗体和抗精子抗体的水平均显著高于对照组。结论抗子宫内膜抗体和抗精子抗体检测在复发性自然流产和原发性不孕患者中具有相关的临床价值,应加强备孕人群的筛查工作,及早干预。     

Association between infertility and spontaneous abortion.

Women experiencing recurrent spontaneous abortion have a higher frequency of infertility than that expected in the general population. To further define the relationships between infertility and spontaneous abortion, the obstetrical histories of 43 women with unexplained secondary infertility were evaluated for the frequency of spontaneous abortion. Of the 88 pregnancies studied, 39 (44%) resulted in spontaneous abortion. Women with unexplained secondary infertility experienced a three-fold increase (P less than 0.0001) in the frequency of spontaneous abortions and half the number of live births (P less than 0.0001) compared with the general population. We conclude that the association between infertility and spontaneous abortion includes a higher frequency of spontaneous abortion among infertile couples as well as a higher prevalence of infertility among recurrent spontaneous aborters compared with the general population.     

Possible susceptibility of the HLA-DPB1*0402 and HLA-DPB1*04 alleles to unexplained recurrent abortion: analysis by means of polymerase chain reaction-restricted fragment length polymorphism method

PROBLEM: To clarify whether HLA-DP antigens are associated with patient population of unexplained recurrent abortion. METHOD OF STUDY: The frequency of HLA-DPB1 alleles in patients with unexplained recurrent abortion, and the compatibility of HLA-DPB1 alleles between patient couples, were studied using a polymerase chain reaction (PCR)-restricted fragment length polymorphism (RFLP) method. Thirty patients who had a history of unexplained primary recurrent abortion, and their husbands, were typed for HLA-DPB1 genotype. Two hundred and ninety-nine base pair fragments from the second exon of HLA-DPB1 genes were selectively amplified using the PCR-primers. After amplification, the DNAs were digested with restriction endonucleases, and subjected to electrophoresis in a 12% polyacrilamide gel to determine HLA-DPB1 genotype. RESULTS: The frequency of HLA-DPB1*0402 and DPB1*04 alleles in the patient group (n = 30) was significantly increased, as compared to that in the normal fertile women (n = 30). The frequency of HLA-DPB1*04 allele in the patient group was significantly increased, as compared to that in the general population (n = 112). No significant compatibility of HLA-DPB1 alleles could be observed between patient couples and normal fertile couples. CONCLUSION: These findings suggest a possible new class II association with patient population of unexplained recurrent abortion.     

Recurrent pregnancy loss and its relation to FV Leiden, FII G20210A and polymorphisms of plasminogen activator and plasminogen activator inhibitor

Thrombophilic disorders and hypofibrinolysis were demonstrated to be risk factors in a majority of women with recurrent pregnancy loss (RPL) and infertility. We investigated the association of FV G1691A mutation, F II G20210A gene polymorphism (PM), 4G/5G PAI-1 and Alu I/D tPA PM in 32 women with infertility and 49 women with at least 2 unexplained early abortions. FV Leiden mutation was significantly more common in women with RPL (10%, p = 0.02) and infertility (19%, p = 0.0005) compared with controls (2%). PAI-1 4G PM and t-PA Alu I PM, alone or in combination, were not associated with RPL or infertility. 9/49 women with RPL showed coagulation disorders with heterozygous FV Leiden mutation (5), FXII (1), protein C (1) or protein S (2) deficiency. However, due to the small number of patients studied, no definite conclusion can be drawn.     

The value of GnRH analogue therapy in IVF in women with unexplained infertility

Seventy-six women with unexplained infertility, undergoing in-vitro fertilization and embryo transfer (IVF-embryo transfer), were selected for three different ovulation induction protocols. In group I, induction of ovulation was performed with pure follicle-stimulating hormone/human menopausal gonadotrophin/human chorionic gonadotrophin (pFSH/HMG/HCG). Group II patients were given a combined therapy consisting of a gonadotrophin-releasing hormone (GnRH) analogue, decapeptyl (DTRP6) followed by pFSH/HMG/HCG. In group III, patients underwent two IVF-embryo transfer cycles, serving as their own controls. The initial cycle was induced with pFSH/HMG/HCG while the second was stimulated using decapeptyl/pFSH/HMG/HCG. Significantly higher rates of fertilization, cleavage and pregnancy (P less than 0.001, P less than 0.07, P less than 0.001, respectively) were achieved in group II patients to whom combined GnRH agonists and gonadotrophins were given. Furthermore, among group III patients, no pregnancies occurred during the initial IVF-embryo transfer cycles whereas a 23% pregnancy rate (P less than 0.001) was obtained after GnRH agonist therapy. Our results indicate that the combination of GnRH agonists and gonadotrophins is of value in cases of unexplained infertility. Further, larger studies must be performed before the true efficacy of this mode of therapy can be determined in women with unexplained infertility.     

Efficient typing of copy number variations in a segmental duplication-mediated rearrangement hotspot using multiplex competitive amplification

Local genomic architecture, such as segmental duplications (SDs), can induce copy number variations (CNVs) hotspots in the human genome, many of which manifest as genomic disorders. Significant technological advances have been achieved for genome-wide CNV investigations, but these costly methods are not suitable for genotyping certain disease-associated CNVs or other loci of interest in populations. Recently, two independent studies showed that the murine meiosis expressed gene 1 (Meig1) was critical to spermatogenesis. We found that the human orthologue MEIG1 is flanked by an SD pair, between which non-allelic homologous recombination (NAHR) can cause recurrent CNVs. To study this potential CNV hotspot and its role in spermatogenesis, we developed a new CNV genotyping method, AccuCopy, based on multiplex competitive amplification to investigate 320 patients with spermatogenic impairment and 93 healthy controls. Three MEIG1 duplications (two in patients and one in controls) were identified, whereas no deletion was found. As NAHR results in more recurrent deletions than duplications at a locus, the over representation of recurrent MEIG1 duplications suggests a potential purifying selection operating on this hotspot, possibly via fecundity. We also showed that AccuCopy is an efficient and reliable method for multiplex CNV genotyping.     

自身抗体与不孕及自发性流产关系的探讨

目的检测抗心磷脂抗体(ACA)和抗精子抗体(AsAb)两种自身抗体在不孕及自发性流产患者中存在的情况,并观察应用阿司匹林治疗ACA阳性反复流产患者的临床效果。方法应用酶联免疫吸附(ELISA)法检测150例原发或继发不孕患者(不孕组)、198例自发性流产或有胚胎停育史患者(流产组)及40例正常对照组血清中的ACA及AsAb抗体。对其中53例ACA阳性反复流产患者在孕前一个月或孕早期采用低剂量阿司匹林治疗。结果不孕组及流产组ACA总阳性率分别为48.00%和50.51%,与对照组(7.50%)相比有非常显著性差异(P<0.001);不孕组及流产组AsAb阳性率分别为31.33%和25.25%,与对照组(10.00%)比较亦有显著性差异(P<0.05)。53例经治疗患者活产婴儿48例,妊娠成功率为90.57%。结论ACA和AsAb等自身抗体是导致不孕及自发性流产的免疫学因素之一,应用低剂量阿司匹林治疗ACA阳性反复流产患者是保证其妊娠成功的有效方法。     

Association of the A/G polymorphism at position 49 in exon 1 of CTLA-4 with the susceptibility to unexplained recurrent spontaneous abortion in the Chinese population

PROBLEM: To investigate whether the A/G polymorphism at position 49 in exon 1 of cytotoxic T lymphocyte antigen-4 (CTLA-4) gene, which delivers a negative signal to T-cell activation, confers the susceptibility to unexplained recurrent spontaneous abortion in the Chinese population. METHOD OF STUDY: A total of 168 patients with unexplained recurrent spontaneous abortion (RSA), who were treated in the Renji Hospital affiliated to the Shanghai Second Medical University, were matched against 117 women with normal pregnancy history. Case-control study to compare the frequency of G/A alleles, AA/AG/GG genotypes and A + (AA + AG) /G+ (GG + AG) phenotypes of CTLA-4 between RSA patients and controls were performed. After amplification of CTLA-4 exon-1 region by polymerase chain reaction (PCR), restriction fragment-length polymorphism (RFLP) was used to detect the polymorphism at position 49 in exon-1 of CTLA-4 gene. Statistical significance was tested by SPSS software. RESULTS: There were dissimilar distributions of G/A alleles, AA/AG/GG genotypes and A+/G+ phenotypes of CTLA-4 between RSA patients and controls. The frequencies of G allele (P = 0.032) and GG genotype (P = 0.011) in RSA patients were significantly higher than those in controls, while the frequencies of AG genotype (P = 0.039) and A + (AA + AG) phenotype in RSA patients were decreased significantly (P = 0.011). CONCLUSIONS: Our findings suggest that A/G polymorphism in exon-1 of CTLA-4 is associated with the immunopathogenesis of RSA, and it confers susceptibility to RSA in Chinese population.     

Lymphoid tissue in the endometrium of women with unexplained infertility: morphometric and immunohistochemical aspects

The purpose of this study was to investigate whether the endometriumof women with unexplained infertility differs in some immunologicalaspects from the endometrium of normal fertile women. Endometrialbiopsies were obtained from 24 normal fertile women (group I)and 24 women suffering from unexplained infertility (group II)at 4, 7, 10 and 13 days following the luteinizing hormone (LH)surge. Endometrial granulated lymphocytes were assessed morphometricallyin 2µm resin sections. A panel of 11 monoclonal antibodieswas employed to characterize the leukocyte subsets in frozensections. Semi-quantification was performed with a Quantimet970 image analyser. Data were analysed using one-and two-wayanalysis of variance. Compared with fertile controls, womenwith unexplained infertility had significantly lower numbersof CD8+ (T suppressor/cytotoxic) cells at each post-LH date.In contrast, the number of CD4+ (T helper/inducer) cells wassignificantly higher in group II. Throughout the luteal phase,infertile women had fewer CD56+ cells than normal fertile controls.The volume fraction of endometrium occupied by the nuclei ofendometrial granulated lymphocytes did not alter with the cyclestage but the mean nuclear diameter and axial ratio decreasedfrom LH+7 to LH+13. The differences observed in endometrialleukocytic subpopulations between fertile and infertile womenmay contribute to unexplained infertility probably by affectingthe embryonic maternal dialogue during the implantation andearly placentation period.     

The prevalence of polycystic ovaries in women with infertility.

Polycystic ovaries (PCO) are highly prevalent in women presenting with hirsutism or recurrent miscarriage but the functional significance of PCO in ovulatory women presenting with infertility remains unclear. We examined the prevalence of PCO, on ultrasonography, among women presenting with infertility. Among 289 couples classified in four main diagnostic categories, PCO were found in 81 (83%) of 98 anovulatory patients, 40 (53%) of 76 patients whose partners had sperm dysfunction, 26 (50%) of 52 patients with tubal disease and in 28 (44%) of 63 patients with unexplained infertility. By comparison, in a control group of 67 parous volunteers, 19 (28%) were found to have PCO. PCO patients with unexplained infertility had higher midfollicular luteinizing hormone and testosterone compared with the group with normal ovaries. The prevalence of PCO was significantly higher in each of the infertility groups than in controls, and a similar tendency (not significant) was observed among women with unexplained infertility. Ovulatory PCO women with infertility had higher testosterone concentrations in comparison with PCO controls. In summary, the prevalence of PCO among ovulatory women with infertility is higher than that in the normal population, suggesting that PCO may, perhaps by virtue of an effect of hyperandrogenaemia, contribute to the causes of subfertility in women with regular menses.     

In Vitro Suppression of Murine Blastocysts Growth by Sera From Women With Reproductive Disorders

ABSTRACT: Early mouse embryos at the two-cell stage were cultured in medium supplemented with sera from women with primary and secondary multiple spontaneous abortions and with long term unexplained infertility as compared to sera obtained from normal fertile women and pooled human male sera. On the basis of microscopic observation and uptake of ³H-thymidine we report a relationship between reproductive histories and the presence of a serum embryo inhibition factor in eight of ten sera samples from women with primary habitual abortions, six of ten sera from women with secondary habitual abortions, and ten of ten sera from women with unexplained infertility. This activity occurs independently of positive maternal antipaternal lymphocytotoxicity. Fractionation of serum samples by ammonium sulphate precipitation, resulted in removal of the embryo-inhibition factor with the IgG fraction in four of five primary habitual abortion cases and in two of five secondary habitual abortion patients, but not in the case of unexplained infertility. We propose that the appearance of such inhibition factor may be of relevance in the etiologies discussed in this paper and may possibly provide the basis for a new classification of idiopathic spontaneous habitual abortions, i.e. positive or negative for the embryo inhibition factor.     

Natural cycle IVF in unexplained, endometriosis-associated and tubal factor infertility.

BACKGROUND: To elucidate possible differences between unexplained and minimal peritoneal endometriosis-associated infertility, we studied their outcome in natural cycle IVF (NIVF). METHODS: A prospective cohort study was carried out on unexplained (33 couples), minimal peritoneal endometriosis-associated (30 couples) and tubal factor (24 couples) infertility in 223 NIVF cycles, using human chorionic gonadotrophin (HCG) for ovulation induction. RESULTS: During the first NIVF attempt, follicular and luteal phase oestradiol, FSH, LH and progesterone concentrations, as well as endometrial thickness and follicular diameter were similar among the three groups. Periovulatory follicular growth monitored from day of HCG administration to oocyte aspiration was significantly lowered in unexplained infertility compared with minimal endometriosis-associated and tubal factor infertility. The fertilization rate, clinical pregnancy rate per initiated cycle, per successful oocyte retrieval and per embryo transfer, in minimal endometriosis (80.0, 10.4, 16.0 and 23.5% respectively) were similar to that in tubal factor infertility patients (68.6, 5.8, 11.4 and 16.0%) but significantly higher (P < 0.05) than that of the unexplained infertility group (62.2, 2.6, 5.4 and 8.7%). CONCLUSIONS: The significant reduction in follicular periovulatory growth, fertilization and pregnancy rates in unexplained infertility compared with minimal peritoneal endometriosis patients may be explained by sub-optimal follicular development with possibly reduced oocyte quality, intrinsic embryo quality factors or by impaired implantation. From a clinical point of view, NIVF is less suited to unexplained infertility treatment, but might represent an interesting treatment option for minimal peritoneal endometriosis-associated infertility.     

叶酸代谢酶基因多态性与反复自然性流产易感性的研究

目的探讨叶酸代谢相关酶基因多态性在不明原因反复自然流产遗传易感性中的作用地位。方法运用聚合酶链反应-限制性片段长度多态性技术（PCR—RFLP）检测蛋氨酸合成酶还原酶（MTRR）A66G,蛋氨酸合成酶（MS）A2756G,N5,10-亚甲基四氢叶酸还原酶（MTHFR）C677T,胱硫醚β-合成酶（CBβS）844ins68基因多态性。结果不明原因反复自然流产患者MTHFR的T等位基因突变频率较正常对照组明显升高。而MS、MTRR和CBβS突变频率在病例组与对照组之间无显著差异。结论MTHFR C677T基因突变多态性可作为不明原因反复自然流产预后的检测指标。     

The production of leukaemia inhibitory factor by human endometrium: presence in uterine flushings and production by cells in culture

The concentration of leukaemia inhibitory factor (LIF) was measured in uterine flushings obtained from normal fertile women, from women with unexplained infertility and from women who suffered recurrent miscarriage. In normal fertile women, LIF was not detected in flushings obtained on days luteinizing hormone (LH)+0 to LH+6 of the cycle, but concentrations gradually increased from day LH+7 to a maximum at day LH+12. The amount of LIF in flushings obtained from women with unexplained infertility was significantly lower than in those from normal fertile women on day LH+10 (P < 0.05). The production of LIF by cultured human epithelial and stromal cells was also investigated. LIF was not detectable in the supernatants of cultured stromal cells. Basal LIF production by epithelial cells varied according to the stage in the cycle at which the biopsy was taken. Significantly more LIF was produced by epithelial cells from late proliferative and early secretory endometrium compared with amounts produced by cells from early proliferative (P < 0.001) and late secretory (P < 0.01) endometrium. High doses of progesterone and oestradiol caused a small decrease in epithelial cell LIF production: the combined effect of progesterone and oestradiol (P < 0.01) was greater than the effect of either steroid alone (P < 0.05). The results show, for the first time, the capability of human endometrium to produce LIF in vivo. The fact that maximum LIF concentrations are present at implantation and that decreased concentrations occur in women with unexplained infertility suggest the importance of this cytokine in embryo implantation.      

原因不明复发性流产患者主动免疫治疗后调节性T细胞比例变化及意义

目的探讨原因不明复发性流产（URSA）主动免疫治疗后外周血CD4＋CD25＋调节性T细胞比例变化及其意义。方法反复流产3～6次的不孕患者（n=55）,采用补体依赖细胞毒实验检测封闭抗体的水平,应用患者丈夫外周血淋巴细胞为患者做皮下免疫治疗,并随访妊娠结局;用双荧光标记流式细胞分析技术检测55名原因不明复发性流产患者治疗前后外周血CD4＋CD25＋调节性T细胞水平的变化。结果55例原因不明复发性流产患者检测封闭抗体阴性后接受主动免疫治疗,其中已分娩41例,14例患者再次流产。主动免疫治疗后,原因不明复发性流产患者外周血CD4＋CD25＋调节性T细胞的比例较治疗前明显增加（P（0.05）;妊娠成功患者外周血CD4＋CD25＋调节性T细胞的比例显著多于妊娠失败者。结论主动免疫对于原因不明复发性流产患者是一种有效的治疗方法;URSA的发生与CD4＋CD25＋调节性T细胞水平降低有关。     

早期不明原因反复自然流产发生与蜕膜中免疫细胞的研究进展

人类妊娠被认为是一种半同种异体抗原移植，母胎间存在着某种免疫耐受机制来维持妊娠的进行，但目前为止这种免疫耐受机制尚不明确。大量的研究发现不明原因反复自然流产患者蜕膜中调节性T细胞的数量和功能均显著降低，表明调节性T细胞在避免胎儿免疫排斥中发挥着重要的作用。同时NK细胞作为早期妊娠蜕膜中的优势淋巴细胞亦对妊娠的维持起着重要的作用，不明原因反复自然流产患者蜕膜NK细胞数量和活性比正常妊娠妇女明显升高，同时CD56^＋CD16^＋／CD56^＋CD16^-NK细胞比例失衡。由此可见，妊娠早期不明原因反复自然流产的发生与蜕膜中淋巴细胞的异常表达相关，通过对这种复杂机制的研究可以为不明原因反复自然流产的预防和治疗提供依据。