Immune cells contribute to systemic cross-talk between the embryo and mother during early pregnancy in cooperation with the endocrine system

In early pregnancy, human chorionic gonadotropin (HCG) stimulates the corpus luteum to produce progesterone that in turn maintains human embryo implantation in the uterus. This inevitable communication through blood circulation can be called 'systemic cross-talk between the embryo and mother'. Despite considerable evidence suggesting that the human corpus luteum cannot be maintained by HCG alone, no other responsible soluble factors have been proposed. We found that peripheral blood mononuclear cells (PBMC) derived from pregnant women promoted progesterone production by human luteal cells and propose that both hormones and immune cells participate in this systemic cross-talk. This systemic cross-talk by immune cells is believed to operate in embryo implantation. Splenocytes derived from pregnant mice promoted endometrial differentiation and embryo implantation in vivo . Human PBMC derived from women early in pregnancy promoted invasion of murine embryos in vitro. In addition, recombinant HCG increased the effects of human PBMC on murine embryo invasion. Human chorionic gonadotropin also increased chemokine production by human PBMC through a lectin–glycan interaction, which is a primitive pathway in the immune system. Furthermore, chemokines were shown to induce human trophoblast invasion. These findings suggest that the immune system positively contributes to systemic cross-talk between the embryo and mother in cooperation with the endocrine system. (Reprod Med Biol 2006; 5 : 19–29)     

Administration of gonadotropins stimulates proliferation of normal mouse ovarian surface epithelium.

Little is known concerning the proliferation of the ovarian surface epithelium or the factors which control this process. To define when and under what circumstances this epithelium proliferates, we have studied the proliferation of mouse ovarian surface epithelium (OSE) during embryogenesis, early postnatal life, various physiological circumstances in the adult and in response to gonadotropic hormones, using the bromodeoxyuridine technique. Proliferation of the OSE is greatest during embryonic development, and falls gradually after birth until sexual maturity is reached. Very little proliferation of the OSE is detectable in adult life in non-pregnant, pregnant or lactating mice. The basal proliferation of the OSE can be increased significantly by inducing follicular development with pregnant mare serum gonadotropin (PMSG) or by administration of the pure recombinant gonadotropins follicle-stimulating hormone (FSH) or luteinizing hormone (LH). These results show that administration of gonadotropins to sexually mature mice induces proliferation of ovarian surface epithelium concurrently with the process of folliculogenesis.     

Optimization of superovulation in the reproductively mature mouse

Optimum gonadotropin doses and chronology were established for the induction of superovulation in sexually mature hybrid mice (BALB/cBy×C57BL/6By). A regime of 12 IU pregnant mares' serum gonadotropin (PMSG), followed 48 hr later by 20 IU human chorionic gonadotropin (hCG) administered 1 hr before the midpoint of the light cycle (1200), gave the maximum ovulatory response. There was no evidence that endogenous luteinizing hormone influenced the superovulation response to exogenous gonadotropins. Fewer than 50% of zygotes reached the blastocyst stage (90–93 hr post hCG), with the greatest rate of loss at the two-to four-cell stage. Litter size following superovulation was 19.6±0.9. There was no significant difference between the number of blastocysts observed and litter size. Similarly, counts of mature follicles in ovaries prior to hCG stimulation were not significantly greater than the number of secondary oocytes that subsequently ovulated. These data indicate that standard superovulation protocols may require finetuning to maximize productivity and confirm that embryo loss is greatest between the first cleavage division and blastocyst formation.     

Hormonal regulation of galectin 3 in trophoblasts and its effects on endometrium

Previous studies had shown important functions of galectin 3 (Gal-3) in endometrium during embryo implantation, in regulation of endometrial cell proliferation and adhesion by interacting with integrin β3. In this study, we investigated hormonal regulation of Gal-3 in trophoblasts and its extracellular effects on endometrium. We used BeWo and RL95-2 cells as a model of trophoblastic and endometrial epithelial cells, respectively, to create an in vivo model of embryo implantation. Our results indicated that 17β-estradiol (E2), progesterone (P4), and human chorionic gonadotropin (hCG) induced the expression of Gal-3 and promoted its secretion from BeWo cells. The exogenous Gal-3 inhibited cell proliferation and induced apoptosis of endometrial cells (RL95-2 cells) through activation of integrin β1. We further validated the proapoptotic effect of Gal-3 secreted by trophoblastic cell on endometrial cells by culturing RL95-2 cells with Bewo cells and measuring the apoptotic rate. Our analysis provides new insight into the critical roles of Gal-3 in embryo implantation.     

银杏内酯B对小鼠胚胎着床过程中滋养层和蜕膜组织超微结构的影响

目的:观察银杏内酯B(GB)抗小鼠胚胎着床的组织形态学与超微结构变化特征。方法:将25只未经产成年雌性昆明小鼠随机分成5组,每组5只,于妊娠第2日右侧子宫角注射不同剂量的GB(2 mg/kg、4 mg/kg、6 mg/kg、8 mg/kg、10 mg/kg)作为实验侧,左侧子宫角注射等量的生理盐水作为阴性对照侧。另取3只孕鼠不作任何处理,作为空白对照组。于妊娠第8日处死小鼠,取子宫组织,观察GB对滋养层细胞和子宫蜕膜细胞组织形态学和超微结构的影响。结果:妊娠第8日,实验侧小鼠胚胎发育不良,子宫壁充血,胚胎总数显著少于阴性对照侧。光镜下观察到实验侧蜕膜相对较薄、绒毛间隙有白细胞浸润、巨细胞结构松散,并有部分蜕膜细胞发生变性和坏死;电镜下观察到实验侧滋养层细胞胞质电子密度减低、出现小空泡、核形态发生改变、蜕膜细胞胞质内溶酶体增多、线粒体发生肿胀,而且随着GB剂量增大(≥8 mg/kg),滋养细胞胞质出现大量溶酶体、核固缩、碎裂,蜕膜细胞胞质亦出现大量溶酶体、核固缩、碎裂,并出现核膜溶解。结论:GB可造成绒毛滋养细胞及蜕膜细胞超微结构的改变,破坏胚胎赖以生存与发育的内环境,从而干扰胚胎着床。     

TRAIL在小鼠胚胎着床过程中子宫内膜的表达

目的:检测TRAIL在小鼠胚胎着床过程中子宫内膜的表达,探讨它在蜕膜细胞凋亡中的作 用。方法:采用RT-PCR及免疫组化技术检测妊娠d 1-8小鼠子宫组织TRAILmRNA及蛋白的表 达情况。结果:妊娠d 1-8的小鼠子宫组织均有TRAIL mRNA的表达,且着床期间的表达较着床前 明显增加(P<0．05)。妊娠d 1-3,小鼠子宫内膜无TRAIL蛋白表达;妊娠d 4,TRAIL表达在小鼠胚 胎定位、黏附点的子宫内膜腔上皮细胞;妊娠d 5-6,TRAIL定位于胚胎着床点附近的蜕膜细胞中; 妊娠d 7-8,TRAIL表达在与子宫蜕膜邻近的胚胎滋养层细胞中。结论:在小鼠胚胎着床过程中, TRAIL诱导子宫内膜腔上皮细胞凋亡可能是胚胎跨越上皮屏障的重要机制之一,且TRAIL诱导的 蜕膜细胞和胚胎滋养层细胞的凋亡在滋养层细胞对子宫内膜的适度侵入过程中起重要作用。     

Perigestational alcohol consumption induces altered early placentation and organogenic embryo growth restriction by disruption of trophoblast angiogenic factors

Research questionMaternal alcohol consumption produces fetal retardation and malformations, probably associated with placental defects. Does perigestational alcohol consumption up to organogenesis lead to abnormal placentation and embryo growth restriction by disrupting the vascular endothelial growth factor (VEGF) system in embryo–placental development?DesignFemale mice were treated with 10% ethanol in drinking water before and up to day 10 of gestation. Control mice received ethanol-free water. After treatment, the trophoblastic tissue, embryo growth and the angiogenic VEGF pathway were analysed.ResultsFemale mice who had received treatment had resorbed and delayed implantation sites with poor ectoplacental cone development. Reduced trophoblastic area tissue from female mice who had received treatment had abnormal junctional zone and diminished labyrinthine vascularization. After treatment, the labyrinth had increased chorionic trophoblast proliferation, hypoxia inducible factor-1α immunoexpression but reduced apoptosis. The embryo growth was reduced concomitantly with low VEGF immunostaining but high endothelial nitric oxide synthase (eNOS) expression. In junctional and labyrinth of treated female mice, gene and protein immunoexpression of VEGF was reduced and the protein expression of FLT-1 increased compared with controls. Increased activation of kinase insert domain receptor receptor (phosphorylated KDR) and expression of eNOS were observed in placenta of treated female mice. Immunoexpression of metalloproteinase-9, however, was reduced in junctional zone but increased in labyrinth, compared with controls.ConclusionsThese data reveal inadequate expression of VEGF/receptors and angiogenic eNOS and metalloproteinase factors related to abnormal early placentation after perigestational alcohol ingestion, providing insight into aetiological factors underlying early placentopathy associated with intrauterine growth restriction caused by maternal alcohol consumption.     

Transvaginal ultrasound-guided embryo transfer improves outcome in patients with previous failed in vitro fertilization cycles

OBJECTIVE: To determine the effect of transvaginal ultrasound-guided ET in IVF cycles performed on patients who had previously failed to conceive from IVF and compare the results to previous cycles where ultrasound guidance was not used. DESIGN: Retrospective clinical study.Setting: Private practice IVF program. PATIENT(S): One hundred twenty-nine women undergoing consecutive cycles of IVF where fresh embryos were transferred. INTERVENTION(S): Transvaginal ultrasound guidance was used during transfer of embryos. MAIN OUTCOME MEASURE(s): Patient age, number of ampules of gonadotropin used, maximum E(2) level, number of oocytes retrieved, number of two pronuclei embryos obtained, number of embryos transferred, mean embryo score, implantation and pregnancy rate. RESULT(S): There was no difference in any of the clinical parameters measured in IVF cycles resulting in pregnancy when transvaginal ultrasound-guided ET was used compared to the failed cycles when there was no ultrasound guidance. Of the patients who previously had failed IVF cycles and subsequently had IVF cycles with ultrasound guidance, those who became pregnant had higher mean embryo scores than those who did not become pregnant. Overall implantation and pregnancy rates were higher during the study period when transvaginal ultrasound guidance was used than in the previous 3 years when it was not used. CONCLUSION(S): Transvaginal ultrasound-guided ET may be responsible for successful IVF cycles in patients who had previously failed to conceive when embryos were transferred by the clinical touch method. Transvaginal ultrasound guidance may also be responsible for an overall increase in embryo implantation and pregnancy compared to the use of the clinical touch method.     

Ovarian hyperstimulation inhibits embryo implantation in the mouse

Embryo implantation is dependent on the synchronous development of the embryo and of the endometrium. Pharmacologic doses of estrogens change endometrial histology and are known to inhibit implantation. During controlled ovarian hyperstimulation, such as occurs during an in vitro fertilization cycle, serum estradiol levels may be elevated to as much as three to six times those found during spontaneous cycles. Serum progesterone levels are also increased and may counteract the elevated estradiol levels. The overall effect of ovarian stimulation on implantation is therefore not known. To study this question, we developed a mouse embryo donation model. Donor embryos were obtained in the late morula to early blastocyst stage from hyperstimulated mated mice. The donated embryos were then transferred to the uteri of two groups of recipient mice. The study group underwent ovarian hyperstimulation with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) (OHR group), while the controls were allowed to cycle spontaneously (SR group). All recipient mice underwent cervical stimulation to induce a pseudopregnant state. Five embryos were transferred to the left uterine horn of each of nine OHR mice and seven SR mice. A higher implantation rate was noted in the SR group than in the OHR group (50±12 vs 8±4%, P<0.001). Our data suggest that, in the mouse, ovarian hyperstimulation impedes implantation by causing adverse changes in uterine receptivity.     

Ovarian stimulation protocol for in vitro fertilization with gonadotropin-releasing hormone agonist widens the implantation window

Pregnancy rates vary considerably with the type of ovarian stimulation used for in vitro fertilization and embryo transfer (IVF-ET). The window of implantation may represent one of the rate-limiting steps in IVF success. We therefore investigated estimated implantation times of 10 consecutive IVF singleton pregnancies, achieved using pituitary suppression with gonadotropin-releasing hormone agonist (GnRH-a) before and during ovarian stimulation with human menopausal gonadotropins (hMG), and compared those with 9 consecutive IVF pregnancies achieved by hMG stimulation only. Estimated implantation times were calculated by regression analysis of serial human chorionic gonadotropin (hCG) measurements between days 7 and 16 after ET. The GnRH-a/hMG pregnancies implanted between days 7 and 11, whereas hMG pregnancies implanted between days 7 and 9 after ET. The hCG regression curve for the GnRH-a/hMG pregnancies revealed a delay of 1.5 days in estimated implantation time compared with the hMG only group. There were no significant differences in pretransfer in vitro embryos development between the two groups. Thus, the delay in hCG rise probably reflects a delay in embryo implantation. We therefore conclude that a GnRH-a/hMG stimulation protocol appears to widen the implantation window in comparison with a hMG only protocol. This observation may at least in part explain the improved IVF pregnancy success with GnRH-a/hMG stimulation protocols.     

Delayed embryo implantation following in vitro fertilization and embryo transfer (IVFET)

Purpose: Our goal was to compare serum human chorionic gonadotropin (hCG) levels in singleton pregnancies achieved following IVFET with those achieved following spontaneous conception. Results: The mean serum hCG level of patients who became pregnant following IVFET lagged 1.5 days behind that of patients who became pregnant spontaneously. Conclusions: The use of gonadotropin releasing hormone analogue as part of the stimulation protocol leading to egg retrieval and IVFET results in a delay in embryo implantation.     

Effects of functional ovarian cysts detected on the 7th day of gonadotropin-releasing hormone analog administration on the outcome of IVF treatment

OBJECTIVE: To investigate the impact of functional ovarian cysts on the time required to achieve pituitary suppression, follicular development, embryo quality, and pregnancy rates during IVF treatment. DESIGN: Prospective observational study. INTERVENTION(S): Daily treatment with buserelin (sc 500 microg) was initiated on day 2 of menstruation. Ultrasound and hormonal tests were performed on days 1, 7, 11, 14, and weekly thereafter until pituitary suppression was achieved. RESULT(S): 48 patients underwent 51 cycles of IVF treatment. A functional cyst was detected in three cycles (5.8%) with baseline ultrasound scan and in 27 cycles (52.9%) on day 7 of buserelin administration. Patients who developed a cyst required a significantly longer time to achieve pituitary suppression (21 vs. 7 days), had a significantly lower FSH level at the time of initiation of gonadotropins, required more ampules of gonadotropin (45 vs. 41 ampules), developed less follicles (13 vs. 17.5), and had lower embryo quality. However, there were no differences in the implantation (23.5% vs. 17.2%) and pregnancy rates (37.2% vs. 29.2%) between two groups. CONCLUSION(S): Functional cysts prolong the period to achieving pituitary suppression, increase gonadotropin requirements, and decrease follicular recruitment and embryo quality. They have, however, no negative effect on pregnancy rates.     

A correlative study on the embryotrophic property of patient's serum and the outcome of in vitro fertilization of human oocytes

The embryotrophic property of patient's serum previously used for in vitro fertilization (IVF) was studied by culturing postimplantation organogenesis-stage mouse embryos in a medium containing equal parts of Dulbecco's modified Eagle's medium and the patient's serum. Results of embryo culture experiments were generally found to correlate well with the outcome of IVF. Serum of patients whose oocytes were fertilized and developed to cleavage-stage embryos scored highest for embryotrophic parameters such as morphologic score and protein content of the mouse embryo. There was no significant difference in terms of serum embryotrophic activity between patients who became pregnant after embryo transfer and those who did not. When mouse embryos were cultured in serum from cycles with poor IVF results, i.e., oocytes failed to be fertilized or fertilized oocytes failed to cleave or cleave abnormally, significantly retarded embryonic growth and a higher incidence of malformed embryos were observed. However, in two cases where IVF failed as a result of poor semen quality, the patient's serum was found to be supportive of mouse embryonic development.     

Analysis of Implantation in Assisted Reproduction Through the Use of Serial Human Chorionic Gonadotropin Measurements

Purpose: Our purpose was to examine implantation of singleton pregnancies achieved following various assisted reproductive technologies (ARTs) through the appearance and rising titers of serum human chorionic gonadotropin (hCG) levels. Methods: A total of 114 singleton pregnancies resulting from in vitro fertilization and intrauterine insemination was analyzed. Patients were divided into five groups according to the type of ovarian stimulation protocol [gonadotropin stimulation with/without the use of gonadotropin-releasing hormone agonist (GnRHa), long protocol, or flare-up technique] and to the day of embryo transfer (day 2 or day 3 after oocyte retrieval). Serial serum hCG levels were measured between 10 and 25 days after fertilization and log-transformed. Linear regression analyses were performed and extrapolated to hCG = 10 mIU/ml (hCG ₁₀ ), which was used as an estimate of detectable implantation. The slopes of the regression lines were used to estimate the rising speed of hCG. Results: There were no significant differences in the days of hCG in maternal serum to reach 10 mIU/ml (implantation) or in the slopes of the regression lines for all five studied groups. Conclusions: The appearance of hCG in maternal serum was used to assess the time of clinically detectable implantation. Furthermore, because hCG production is a marker of trophoblastic activity, its serum doubling time was used as an indicator of embryo quality. Results showed that in various ART protocols with and without GnRHa, there were no significant differences in implantation time or embryo quality. Embryo development in early pregnancy follows a preprogrammed-timing schedule and depends mainly on the embryonic age of the health, successfully implanted conceptus.     

The efficiency of human reproduction after in vitro fertilization and embryo transfer

Two hundred ninety-seven nonpregnant patients were used to study the possibility of early transient implantation as a parameter of the efficiency of in vitro fertilization (IVF) procedures. Ten patients without embryo transfer (ET) were used as controls. The luteal estradiol, progesterone, and human chorionic gonadotropin were measured by radioimmunoassay (RIA). In 31 cases, a transient elevation of hCG occurred after complete serum clearance of exogenous hCG, suggesting that the transient increase in hCG was of embryonic origin. In addition, five patients were found to have prolonged clearance of hCG, which was due not to individual variation in clearance, but to a minimal production of hCG by trophoblastic tissue. These data suggest that implantations occurred in 12.1% of our so-called "nonpregnant" patients.     

Preimplantation development following in vitro fertilization of mouse oocytes: Effects of timing of superovulation and preincubation in vitro

The early embryonic development of in vitro fertilized oocytes was assessed following superovulation in F₁ hybrid C57BL/6×CBA/Ca mice. Decreasing the time interval between the administration of constant doses of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) resulted in decreases in the frequency of development to the blastocyst stage but had no significant effect on development to the two-cell stage. Preincubation of postovulatory oocytes in vitro prior to insemination did not compensate for the reduced preovulatory development in vivo but resulted in decreases in the frequency of development to the blastocyst stage. The results indicate that inadequate preovulatory development of superovulated mouse oocytes can adversely affect the preimplantation development of in vitro fertilized embryos in the absence of a visible inhibitory effect on development to the two-cell stage and also that preincubation of postovulatory oocytes in vitro prior to fertilization reduces subsequent developmental capacity.     

彩色多普勒超声评价反复种植失败患者子宫动脉血流特点研究

目的 探讨反复种植失败（recurrent implantation failure,RIF）患者子宫动脉血流及其相关因素的特点。方法 选取2012年3月至2013 年8月于烟台毓璜顶医院生殖医学科因输卵管因素行体外受精-胚胎移植（in vitro fertilization- embryo transfer,IVF－ET）治疗的RIF患者（RIF组,n=40）为研究对象;同期IVF－ET治疗首次助孕成功的患者（首次IVF助孕成功组,n=40）,首次IVF－ET助孕治疗失败患者（首次IVF助孕失败组,n=40）为对照组,进行回顾性分析。于注射人绒毛膜促性腺激素（human chorionic gonadotropin,HCG）日行经阴道彩色多普勒超声检查,测量子宫动脉搏动指数（uterine artery pulsatility index,UAPI）、子宫内膜厚度,并同时测定血雌二醇及孕酮值。比较三组间相关指标的差异。结果 RIF组患者UAPI 3.3±0.4,显著高于同期IVF-ET治疗首次助孕成功组2.3±0.5及失败组UAPI 2.4±0.4,差异有统计学意义（P<0.05）,而IVF-ET治疗首次助孕成功组与失败组相比,差异无统计学意义（P＞0.05）。3组间的子宫内膜厚度、雌二醇及孕酮值差异无统计学意义（P>0.05）。结论 HCG日测定UAPI有助于评价子宫内膜容受性,预测种植率。     

Human implantation: the last barrier in assisted reproduction technologies?

Implantation processes are highly complex involving the actions of numerous hormones, immunoglobulins, cytokines and other factors in the endometrium. They are also essential matters for the success of assisted reproduction. The nature of early embryonic development is of equal significance. It involves ovarian follicle growth, ovulation, fertilization and preimplantation growth. These processes are affected by imbalanced chromosomal constitutions or slow developmental periods. Post-implantation death is also a significant factor in cases of placental insufficiency or recurrent abortion. Clearly, many of these matters can significantly affect birth rates. This review is concerned primarily with the oocyte, the early embryo and its chromosomal anomalies, and the nature of factors involved in implantation. These are clearly among the most important features in determining successful embryonic and fetal growth. Successive sections cover the endocrine stimulation of follicle growth in mice and humans, growth of human embryos in vitro, their apposition and attachment to the uterus, factors involved in embryo attachment to uterine epithelium and later stages of implantation, and understanding the gene control of polarities and other aspects of preimplantation embryo differentiation. New aspects of knowledge include the use of human oocyte maturation in vitro as an approach to simpler forms of IVF, and new concepts in developmental genetics.