Antibody reactivity with porcine zona pellucida

A comparative study on anti-zona antibody activities in the sera from clinically defined categories of patients registered at the WHO Reference Bank for Reproductive Immunology was performed at five different laboratories with different detection methods. Considerably higher incidences of positive reactions were detected by immunofluorescence on porcine zonae in infertile women (16.3%) than in control subjects (7.1%). A similar proportion of positives was found by radioimmuno-binding assay (RIBA) using porcine zona antigen preparation in the infertile group (13.0%) but not in the female control group (0%), giving an indication of the specificity of this test. It is noteworthy that high incidences of positives were observed by RIBA with sera from male subjects with unexplained sterility, vasectomy and aspermatogenesis. A test system of passive hemagglutination reaction (PHAR) using purified porcine zona substance as antigen gave a low but slightly higher incidence of positives in infertile (3.1%) than in control sera (0.9%). No positive reactions were observed with infertile and control sera by another PHAR or by radioimmunoassay using an antigen preparation common to the two test systems. Anti-zona activities in these sera were therefore seen to vary, depending largely upon the detection systems and the antigen preparations.     

Monoclonal antiphospholipid antibody reactivity against human placental trophoblast.

Naturally occurring antibodies against the negatively charged phospholipids cardiolipin (CL) and phosphatidylserine (PS) have been associated with recurrent pregnancy loss. One prevalent hypothesis proposes that antiphospholipid antibody (aPL) mediated pathophysiology is through increased placental thrombosis. In this study we investigated the reactivity of three mouse monoclonal aPLs with term and 26 week human placental preparations. Each monoclonal antibody reacted differently with CL and PS; 3SB9b reacted with PS (CL-/PS+), D11A4 reacted with CL (CL+/PS-) and BA3B5C4 reacted with both CL and PS (CL+/PS+). 3SB9b reacted strongly with the syncytiotrophoblastic layer of both formalin fixed and frozen placental tissue. Sporadic reactivity was observed against the cytotrophoblastic layer. BA3B5C4 reacted strongly and specifically with cytotrophoblastic cells. D11A4 had only weak reactivity in the subtrophoblastic stromal region of the placenta in frozen sections. aPL staining was also observed against extravillous cytotrophoblast. BA3B5C4 stained cytoplasmic structures, whereas 3SB9b stained the plasma membrane region with little cytoplasmic staining. These data suggest that the trophoblastic layer is reactive with aPLs and may potentially be directly damaged through mechanisms unrelated to thrombosis. In addition, the trophoblastic layer directly in contact with the maternal circulation is most reactive with aPLs that are PS+ rather than CL+. The differential reactivity of 3SB9b and BA3B5C4 suggests that the antigenic conformation involving PS on the cytotrophoblast is altered concurrent with fusion into the syncytium.     

Liver X receptors mediate inhibition of hCG secretion in a human placental trophoblast cell line

Liver X receptors (LXR) alpha and beta are important regulators of lipid homeostasis in liver, adipose and other tissues. However, no such information is available for the human placenta. We determined expression of both LXR alpha and beta in placental trophoblast cell lines, BeWo and JAR. Exposure of BeWo cells to a synthetic LXR agonist, T0901317, resulted in an increase in the amount of mRNA of LXR target genes, sterol regulatory element-binding protein-1 and fatty acid synthase. T0901317 also increased the synthesis of lipids. Moreover, T0901317 resulted in a reduced secretion of hCG during differentiation of these cells. Our data for the first time demonstrate a new role for LXRs in the human placenta.     

Antiphospholipid antibodies prevent extravillous trophoblast differentiation

OBJECTIVE: We investigated the hypothesis that antiphospholipid antibodies (aPL) have a detrimental effect on human extravillous trophoblast (EVT) differentiation into giant multinucleated cells "in vitro." DESIGN: The EVT were isolated from the placental chorion using enzymatic digestion and Percoll gradient centrifugation. After 24, 36, and 48 hours in culture, giant multinuclear cells (GMC) were identified by immunohistochemistry using antibodies to cytokeratin 7 and counted. SETTING: An academic research laboratory. PATIENT(S): Placentas were donated by women having an elective cesarean section for a normal pregnancy at term. MAIN OUTCOME MEASURE(S): This model was then used to investigate the effect of two different monoclonal aPL to beta2-glycoprotein 1 (IIC5 and ID2), and control mouse IgG antibody on EVT differentiation. RESULT(S): Freshly isolated EVT were nonproliferative but moved together losing their intervening cell walls and differentiated into GMC. Maximal numbers of GMC were detected after 48 hours of culture. The aPL, IIC5, and ID2 significantly inhibited GMC formation, whereas the mouse IgG control had no effect. CONCLUSION(S): Antiphospholipid antibodies can inhibit EVT differentiation and GMC formation "in vitro" suggesting that a failure of trophoblast differentiation and subsequent uteroplacental development may be an underlying pathology in antiphospholipid syndrome-associated pregnancy loss.     

Persistent low levels of serum hCG due to heterophilic mouse antibodies: an unrecognized pitfall in the diagnosis of trophoblastic disease

Phantom hCG refers to persistent mild elevations of hCG, leading physicians to unnecessary treatments whereas neither a true hCG nor a trophoblastic disease is present. We report the case of a 23-year-old woman with persistent low levels of serum hCG detected one month after miscarriage. As choriocarcinoma was suspected, a chemotherapy trial of methotrexate was prescribed, without any hCG reduction. Subsequently, laparoscopy ruled out a trophoblastic residue and the patient was referred to the Endocrine Unit for further investigations. While low levels of hCG were still detected in serum, no hCG was detected in the urine. In addition, when serum was processed in a HBT tube for revealing heterophilic antibodies, hCG was no longer detected. Such finding indicated the presence of phantom hCG due to heterophilic mouse antibodies interaction. This case raises the need of clinico-biological discussion to avoid inappropriate therapeutic decisions. Based on this case experience and after review of the literature, we suggest that current gynecological protocols for the diagnosis and treatment of trophoblastic disease should consider the inclusion of urinary hCG and/or a test for serum heterophilic antibodies when appropriate.     

Antiphospholipid Antibody Syndrome in Pregnancy

Objective: to provide an overview of the clinical significance of antiphospholipid antibodies (APLA) in pregnancy, dealing mainly with the diagnosis and management of patients with this disorder.Data Sources: sources were identified from a MEDLINE search of English language articles published from 1985 to 1995 (Keywords: lupus anticoagulant, anticardiolipin antibodies, antiphospholipid antibodies, and antiphospholipid antibody syndrome). Additional sources were identified from references cited in relevant research articles.Methods of Study Selection: thirty-six articles where selected, including publications that recorded the prevalence of commonly described antiphospholipid antibodies, clinical significance, laboratory testing, and management of pregnant women with this disorder. Almost all articles were based on case series, meta-analysis, and prospective trial. Management protocol was based on the findings and recommendations of prospective trials and clinical reviews.Results: lupus anticoagulant (LA) and anticardiolipin (ACL) antibodies belong to a heterogeneous group of antibodies directed against protein epitopes that form complexes with negatively charged phospholipids. The selected studies were reviewed critically and their conclusions were evaluated; the available literature indicates that these antiphospholipid antibodies are frequently found at low levels in otherwise healthy people. At higher levels, the presence of these antibodies is closely associated with the occurrence of arterial and venous thromboembolism, thrombocytopaenia, fetal loss, and a variety of other conditions in patients with and without systemic lupus erythematosus (SLE). This combination of significant antiphospholipid antibody levels with certain clinical sequelae defines the presence of antiphospholipid antibody syndrome (APLAS).Conclusions: screening for antiphospholipid antibodies in the prenatal population seems unwarranted. If investigations of women at risk for APLAS are positive, close maternal and fetal surveillance are required. Prophylactic treatment might be used in this select group of patients, although optimal therapy has yet to be defined. Even though knowledge of APLAS pathophysiology is limited, it is currently the guide to treatment options. All prenatal patients with APLAS should receive low dose acetylsalicylic acid (ASA) and either heparin or steroids. Heparin is suggested if there is a history of thrombosis or placental infarction, while steroids should be considered if APLAS is secondary to SLE, or if there is previous evidence of inflammation and complement activation. Defining optimal therapy will require a large multicentre prospective trial. A detailed management protocol is proposed.     

Antibodies to spermatozoa and seminal plasma antigens detected by various enzyme-linked immunosorbent (ELISA) assays

Various ELISA methods have been applied by different research centers to test the efficiency of this approach for the diagnosis of sperm-immune infertility cases. The antigens used were either whole spermatozoa or solubilized spermatozoal membrane preparations and were immobilized on microtiter plates, except in one case where plastic beads were employed. Polyvalent second antibodies or protein-A labelled with enzymes served as tracers. A high frequency of positive sera was found in all groups including fertile controls with tests using whole spermatozoa as antigen. The methods using solubilized antigen preparations showed fewer positives on the whole and correlated better with the various clinical categories of the WHO sera. Whilst there was some agreement in the results between the various laboratories on a few sera, most of the positive sera found by one laboratory were reported as negative by others. More investigative work is needed to improve reproducibility between different laboratories and to reduce non-specific reactions with normal controls. A more precise definition of the proper cut-off levels for positives and negatives is also needed. Despite these short-comings, the development of an ELISA for the diagnosis of sperm-immune infertility cases seems to be justified in the long term.     

Antiphospholid antibodies regulate the expression of trophoblast cell adhesion molecules

OBJECTIVE: To examine the effect of antiphospholipid antibodies on trophoblast expression of adhesion molecules. DESIGN: Primary cytotrophoblast cell cultures. SETTING: Department of Obstetrics and Gynecology, Catholic University, Rome, Italy. PATIENT(S): Five normal pregnant women underwent uncomplicated vaginal delivery at 36 weeks of gestation. INTERVENTION(S): IgG antibodies were isolated from a patient with antiphospholipid syndrome and from a normal control subject, using protein-G Sepharose columns. Cytotrophoblast cells were dispersed in bicarbonate buffer containing trypsin and DNAse I. MAIN OUTCOME MEASURE(S): We investigated the effects of antiphospholipid antibodies on trophoblast adhesion molecules (alpha1 and alpha5 integrins, E and VE cadherins), both at the protein and mRNA levels. RESULT(S): The alpha1 and alpha5 integrins were present in trophoblast cells from 24 hours of culture. Treatment with IgG that were obtained from the patient with antiphospholipid syndrome significantly decreased alpha1 integrin and increased alpha5 integrin at both the mRNA and protein levels. Furthermore, IgG with antiphospholipid antibodies activities induced VE-cadherin down-regulation and the E-cadherin up-regulation at protein and mRNA levels compared with control IgG or untreated cells. CONCLUSION(S): The results suggest that the inadequate trophoblastic invasion, induced by antiphospholipid antibodies, can be the result of abnormal trophoblast adhesion molecules expression.     

Successful pregnancy following low-dose hCG administration in addition to hMG in a patient with hypothalamic amenorrhea due to weight loss

We describe successful ovulation induction with low-dose hCG administration in addition to hMG in a patient with refractory hypothalamic amenorrhea. A 24-year-old woman with weight loss-related amenorrhea underwent ovulation induction and intracytoplasmic sperm injection (ICSI). Administration of exogenous gonadotropins was ineffective in ovulation induction. Supplementation with low-dose hCG in order to increase luteinizing hormone (LH) activity in the late follicular phase produced late folliculogenesis and steroidogenesis, and ovulation was then successfully induced. This report reacknowledges the critical role that LH plays cooperatively with follicle-stimulating hormone in both folliculogenesis and steroidogenesis.     

Luteinizing hormone and human chorionic gonadotropin: distinguishing unique physiologic roles

Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are integral components of the hypothalamic–pituitary–gonadal axis, which controls sexual maturation and functionality. In the absence of signaling through their shared receptor, fetal sexual differentiation and post-natal development cannot proceed normally. Although they share a high degree of homology, the physiologic roles of these hormones are unique, governed by differences in expression pattern, biopotency and regulation. Whereas LH is a key regulator of gonadal steroidogenesis and ovulation, hCG is predominantly active in pregnancy and fetal development. Emerging evidence has revealed endogenous functions not previously ascribed to hCG, including participation in ovulation and fertilization, implantation, placentation and other activities in support of successful pregnancy. Spontaneous and induced mutations in LH, hCG and their mutual receptor have contributed substantially to our understanding of reproductive development and function. The lack of naturally occurring, functionally significant mutations in the β-subunit of hCG reinforce its putative role in establishment of pregnancy. Rescue of reproductive abnormalities resulting from aberrant gonadotropin signaling is possible in certain clinical contexts, depending on the nature of the underlying defect. By understanding the physiologic roles of LH and hCG in normal and pathologic states, we may better harness their diagnostic, prognostic and therapeutic potential.

Chinese abstract

促黄体激素（LH）和人绒毛膜促性腺素(hCG)是下丘脑－垂体－性腺轴整体的组分,控制性成熟和性功能。它们共享受体信号的缺乏,胎儿性分化和出生后发育不能正常进行。尽管它们共享高度的同源性,但这些激素的生理作用是独特的,表达方式、生物效能和调节是不同的,然而,LH 是性腺甾体合成和排卵的关键调节剂,hCG的主要作用是妊娠及孕胎儿发育。新的证据显示以前没有归于HCG的内源性的功能,包括参与排卵和受精,种植,胎盘形成和其他支持妊娠成功的活动。LH,hCG和它们共同的受体自发和诱发突变提供了我们对生殖发展与功能的理解。hCG β-亚单位天然缺乏,功能上明显突变加强了它确定妊娠的公认作用。依据潜在缺陷的本性,因异常促性腺激素信号导致的生殖异常的复苏在某些临床环境下是可能的。通过对LH和hCG 正常和病理状态生理作用的理解,我们可以较好地利用它们的诊断,预测和治疗潜能。     

Timing intrauterine insemination either 33 or 39 hours after administration of human chorionic gonadotropin yields the same pregnancy rates as after superovulation therapy

OBJECTIVE: To compare a short and long interval between hCG administration and IUI after superovulation for the treatment of infertility. DESIGN: Prospective, randomized clinical trial. SETTING: University hospital-based fertility clinic. PATIENT(S): Patients planning superovulation and IUI for the treatment of infertility. INTERVENTION(S): Patients with >or=2 years of infertility enrolled for superovulation and IUI treatment were randomized to IUI after a short (32-34-hour) or long (38-40-hour) interval after hCG injection. Superovulation was accomplished with hMG or recombinant FSH, with dose adjustment until the maturation of two to five follicles, at which time hCG was given. Sperm was prepared with a gradient centrifugation technique, with IUI performed high up in the uterine fundus. MAIN OUTCOME MEASURE(S): Pregnancy rates. RESULT(S): Of the 348 patient cycles randomized, 270 treatment cycles were initiated. Eighty-one initiated cycles were canceled, leaving 189 completed randomized cycles from 75 patients for analysis. Pregnancy rates were not significantly different between groups. There were pregnancies in 20 of the 96 short hCG-IUI interval cycles (21%) and in 14 of the 93 long hCG-IUI interval cycles (15%) (odds ratio = 0.673, 95% confidence interval 0.297-1.518). CONCLUSION(S): Pregnancy rates are the same after superovulation therapy whether IUI is done after a short or a long interval after hCG injection.     

The role of Sep (O-phosphoserine) tRNA: Sec (selenocysteine) synthase (SEPSECS) in proliferation,apoptosis and hormone secretion of trophoblast cells

Objectives

To investigate whether Sep (O-phosphoserine) tRNA: Sec (selenocysteine) synthase (SEPSECS), which plays an essential role in the synthesis of selenoprotein, affects proliferation, apoptosis and hormone secretion of human trophoblast cells.

Methods

Human trophoblast JEG-3 cells were divided into four groups: control group, SEPSECS silenced-expression group, empty vector group and SEPSECS over-expression group. Over-expression and silenced-expression were achieved by transfection with plasmid DNA or RNA oligonucleotide, respectively. 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) and colony formation assays were performed to investigate cell proliferation, while apoptosis was tested by annexin V-FITC, PI double staining and caspases-3 activation assays, enzyme-linked immunosorbent assay (ELISA) was used to determine the level of progesterone (PG) and human chorionic gonadotropin (hCG).

Results

SEPSECS silenced-expression clearly inhibited proliferation of JEG-3 cells (p < 0.05), significantly induced cell apoptosis (p < 0.01) and reduced the production of PG and hCG (p < 0.05). On the contrary, SEPSECS over-expression significantly promoted both cell proliferation (p < 0.01) and secretion of PG and hCG (p < 0.05).

Conclusions

SEPSECS significantly affects proliferation, apoptosis and hormone secretion of human trophoblast cells, suggesting that a potential relationship exists among SEPSECS, cell proliferation, apoptosis and hormone production of human placental trophoblast cells. Furthermore, this may provide a clue to uncover the relationship between selenium and human placental in association with an emphasis on the importance of selenium adequacy during pregnancy.     

Ovarian cancer in infertile women during or after ovulation-inductiontherapy: expression of LH/hCG receptors and sex steroid receptors

Kuroda H, Konishi I, Mandai M, Nanbu K, Rao Ch V, Mori T. Ovarian cancer in infertile women during or after ovulation-induction therapy: expression of LH/hCG receptors and sex steroid receptors. Int J Gynecol Cancer 1997; 7: 451–457.
A possible association between the use of fertility drugs and development of ovarian cancer has recently been suggested. Review of 131 patients with ovarian carcinoma in our hospital revealed that six (4.6%) cases had developed during or after ovulation-induction therapy. To examine the hormone sensitivity of these tumors, the expression of luteinizing hormone/chorionic gonadotropin (LH/hCG) receptors and sex steroid receptors in the tumor cells was analyzed by immunohistochemistry. In addition, to elucidate the clinical and pathological characteristics of ovarian cancer in this setting, all of the 41 cases, including both of our series and previously reported cases in the literature, were reviewed. Immunohistochemical analysis showed that expression of LH/hCG receptors or sex steroid receptors in the tumor cells was observed in five of the six cases. This suggests that gonadotropins and/or sex steroids may influence the biological behavior of the tumor. Ovarian cancer during or after ovulation-induction consisted of two different types: one is serous, low-malignant potential (LMP) or invasive carcinoma and the other is endometrioid or clear cell carcinoma arising in an endometriotic cyst. Although the latter group is usually detected at an early stage, serous tumors are frequently at an advanced stage, even under close examination during infertility treatment. This is essential for giving informed consent during ovulation-induction therapy for infertile women.     

两组生源大学生入学时的血清抗精子抗体检测

目的:了解外招生源和内地生源的大学生入学体检时的血清抗精子抗体的状况。方法:640名大学新生按生源和性别分为4组:外招生男性组96例(A组),女性组115例(B组);内地生男性组256例(C组),女性组173例(D组)。结合新生入学体检获取血清,应用浅盘精子凝集试验(SAT)、浅盘精子制动试验(SIT)和间接免疫珠试验(iIBT),检测血清抗精子抗体。结果:A、B和D组分别检出SAT抗精子抗体阳性各2例,C组检出4例;这10例iIBT的免疫珠附着率为20%-55%。A-D组的SAT抗体阳性率分别为2.1%,1.7%,1.6%和1.2%。B组检出SIT阳性1例。A组与C组、B组与D组的SAT抗体阳性率比较无显著性差异(P>0.05)。结论:外招生源和内地生源的大学生,入学时可检出血清抗精子抗体,需对大学生开展生殖健康和性健康的教育。     

Anti-sperm antibodies, detected by agglutination, immobilization, microcytotoxicity and immunobead-binding assays

To determine the reliability of tests currently utilized in the detection of sperm-reactive antibodies, sera were provided as unknowns and studied without knowledge of the clinical histories. Four laboratories performed tray agglutination tests (TAT), three complement-dependent immobilization (SIT), and single laboratories sperm cytotoxicity (SCT), passive haemagglutination (PHA) and immunobead binding (IBB). Most investigators demonstrated an excellent correlation between duplicate sample results. Nearly all of the female sera were free of anti-sperm antibodies and positive results did not appear in greater frequency in women with unexplained infertility as compared with other categories. For the male sera, the highest incidence of anti-sperm antibodies in the infertile group (21% positive for sperm-reactive IgGs) was obtained by immunobead binding. The GAT and TAT results gave 7 and 12% positives, except for lower results in one laboratory. Sperm-reactive antibodies were detected most commonly in vasectomized men, with all assays except SCT and PHA. Of the newer techniques studied, IBB results correlated well with TAT, GAT and SIT, while SCT and PHA did not, suggesting that a different group of antibodies, perhaps directed against other sperm-associated antigens, was being detected by the latter procedures. In this light, emphasis was placed on the need to validate whether results of particular methodologies correlated with impaired sperm function and to develop methods that provided evidence for this premise, either on the basis of clinical criteria or altered gamete interaction in vitro.     

Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early pregnancy

Problem: There is an increased doubt about the identity of isolated cytotrophoblast cells at term. Therefore, we compared pregnancy serum levels of three hormones [human placental lactogen (hPL), human chorionic gonadotropin (hCG), and leptin] with the capacity for hormone production of early placentae [EP; 8–13 weeks of gestation (WG)] and term placentae (TP; 38–42 WG).Methods: Serum levels of these hormones were determined in 15 paired maternal (7–41 WG) and fetal (37–41 WG) samples. Cytotrophoblast cells were isolated from term (TP; 38–42 weeks) and early (EP; 8–13 weeks) placentae by enzymatic digestion and subsequent purification on a Percoll gradient. These cells were cultured for 6 days. The production of the hormones hPL, hCG, and leptin was determined as release during culture + cell content after culture – cell content before culture.Results: Serum levels (mean ± SD; n ± 15) at 7–12 and 37–41 WG were 89,652 ± 21,431 and 13,620 ± 5854 mIU/ml for hCG, 400 ± 182 and 7088 ± 2030 ng/ml for hPL, and 12,675 ± 4266 and 32,236 ± 10,961 pg/ml for leptin, respectively. For cultured cells from EP and TP, hCG and hPL showed different patterns of release during the first 2–3 days. While the release of these two hormones by EP cytotrophoblast cells continued during 6 days in culture, their concentrations reached a plateau for TP cytotrophoblasts between 4 and 6 days. Leptin was undetectable (<15 pg/ml) in TP cell cultured media, while for EP all three hormones showed the same release profiles. Production calculated for 30,000 TP trophoblast cells cultured for 6 days (n = 8) was 2–31 mIU for hCG and 0.5–2 ng for hPL. For EP (n = 11), it was 50–1070 mIU for hCG, 15–323 ng for hPL, and 137–580 pg for leptin. Net synthesis of hCG and hPL for TP was >10-fold and <1-fold, respectively. In contrast, the production of all three hormones for EP was at least 100 times the initial cell content.Conclusions: These results demonstrate that trophoblasts from early pregnancy show much higher production rates of hCG, hPL, and leptin than at term. However, the in vitro findings are difficult to be reconciled with the different serum concentrations of the two hormones hPL and leptin observed during the course of pregnancy.