Human MLH1/3 variants causing aneuploidy, pregnancy loss, and premature reproductive aging.

Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy loss. Proper disjunction of homologous chromosomes requires the mismatch repair (MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice via genome editing. We identify seven alleles causing reproductive defects in mice including female subfertility and male infertility. Remarkably, in females these alleles cause age-dependent decreases in litter size and increased embryo resorption, likely a consequence of fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that hypomorphic alleles of meiotic recombination genes can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.

Placentation and embryo death in the plains viscacha (Lagostomus maximus).

Caviomorpha are an exceptional group among rodents due to their extended gestational period and the delivery of precocial offspring. Among them, Lagostomus maximus is characterized by its polyovulation, polyembryony, and the highest embryonic death known in mammals. Its chorioallantoic placenta is hemomonochorial, an ancestral character among rodents. It resembles more the human placenta than the murine models. As in all caviomophs, the chorioallantoic placenta is divided in a main placenta and a subplacenta. The former is organized in labyrinth lobes surrounded by trophospongium, as in most caviomorphs. The giant cells (more numerous than in other caviomorphs) near the decidua could be related to invasiveness. During placentation of L. maximus, uterine natural killer cells are found. These cells have been related to invasiveness and remodeling of blood vessels in Mus musculus and Homo sapiens, although in other caviomorphs are not frequently found. In L. maximus, the placenta develops in all conceptuses (5-6 per uterine horn). Necrosis was observed in each implantation site at day 70 post-coitum, except in that closest to the vagina in each horn. This process of embryo death followed by resorption begins at day 26-30 post-coitum. Recently, we found variations in the percentage of blood vessel and uterine gland areas that could explain the regional differences in embryo survival. The characteristics of the placenta and implantation of L. maximus are important to stablish a unique model for studying placentation as well as early embryonic death, of interest for human and veterinary medicine.

Decreased ANGPTL4 impairs endometrial angiogenesis during peri-implantation period in patients with recurrent implantation failure.

Insufficient endometrial angiogenesis during peri-implantation impairs endometrial receptivity (ER), which contributes to recurrent implantation failure (RIF) during in vitro fertilization and embryo transfer (IVF-ET). Angiopoietin-like protein 4 (ANGPTL4) acts as a multifunctional secretory protein and is involved in the regulation of lipid metabolism and angiogenesis in various tissues including the endometrium. Herein, we found decreased ANGPTL4 expression in endometrial tissue and serum during peri-implantation period in 18 RIF-affected women with elevated uterine arterial impedance (UAI) compared with the pregnancy controls. ANGPTL4 and peroxisome proliferator-activated receptor gamma (PPARγ) expression were up-regulated upon decidualization on human endometrial stromal cells (HESCs). Rosiglitazone promoted the expression of ANGPTL4 in HESCs and human umbilical vein endothelial cells (HUVECs) via PPARγ. ANGPTL4 promoted the proliferation, migration and angiogenesis of HUVECs in vitro. Our results suggest that decreased abundance of ANGPTL4 in endometrial tissues impairs the endometrial receptivity via restraining endometrial angiogenesis during decidualization; while rosiglitazone-induced ANGPTL4 up-regulation in hESCs and HUVECs through PPARγ. Therefore, ANGPTL4 could be a potential therapeutic approach for some RIF-affected women with elevated UAI.

Spontaneous embryo resorption in the mouse is triggered by embryonic apoptosis followed by rapid removal via maternal sterile purulent inflammation.

BACKGROUND: In normal mammalian development a high percentage of implantations is lost by spontaneous resorption. This is a major problem in assisted reproduction and blastocyst transfer. Which embryo will be resorbed is unpredictable. Resorption is very fast, so that with conventional methods only final haemorrhagic stages are encountered. Here we describe the histology and immunohistochemistry of 23 spontaneous embryo resorptions between days 7 and 13 of murine development, which were identified by high-resolution ultrasound (US) in a previous study. RESULTS: In the early resorptions detected at day 7, the embryo proper was replaced by maternal haemorrhage and a suppurate focus of maternal neutrophils. In the decidua maternal macrophages transformed to foam cells and formed a second focus of tissue dissolution. In the late resorptions detected at day 9, the embryo underwent apoptosis without involvement of maternal cells. The apoptotic embryonic cells expressed caspase 3 and embryonic blood cells developed a macrophage like phenotype. Subsequently, the wall of the embryonic vesicle ruptured and the apoptotic embryo was aborted into the uterine lumen. Abortion was initiated by degeneration of the embryonic lacunar trophoblast and dissolution of the maternal decidua capsularis via sterile inflammation and accompanied by maternal haemorrhage, invasion of the apoptotic embryo by maternal neutrophils, and contraction rings of the uterine muscle layers. CONCLUSIONS: We conclude that spontaneous resorption starts with endogenous apoptosis of the embryo without maternal contribution. After break down of the foetal-maternal border, the apoptotic embryo is invaded by maternal neutrophils, aborted into the uterine lumen, and rapidly resorbed. We assume that the innate maternal unspecific inflammation is elicited by disintegrating apoptotic embryonic cells.

Maternal nutrient restriction in early pregnancy increases the risk of late embryo loss despite no effects on peri-implantation interferon-stimulated genes in suckler beef cattle.

Reducing feeding costs in suckler beef herds to improve economic returns could have detrimental impacts on fertility. This study sought to determine whether maternal nutrient restriction during early pregnancy affects interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells during the peri-implantation period in two beef cattle breeds. Relationships were also examined between subnutrition and pregnancy failure defined according to ISG fold changes on Days 18 and 21 and to plasma pregnancy specific protein B (PSPB) concentrations on Day 28 post-artificial insemination (AI). Pirenaica or Parda de Montaña dams were assigned to a control (n = 23) or subnutrition (n = 30) group, receiving 100% or 65% of their estimated nutritional requirements from Day 1 to 82 post-AI, respectively. Treatment did not affect ISG expression or fertility. According to ISG fold changes (chi-square P = .023) or PSPB levels (chi-square P = .04) recorded in the subnutrition group, late embryo loss was more likely than in controls. Positive correlation was detected between Day 28 PSPB concentrations and both Day 18 MX1, MX2 and ISG15 expression, and Day 21 OAS1 expression. OAS1 and MX1 fold changes were found to be the best variables to discriminate pregnancy status. Our findings indicate that maternal nutrient restriction during the first third of pregnancy does not impair embryo signalling yet may increase the risk of pregnancy failure.

High-resolution contrast-enhanced microCT reveals the true three-dimensional morphology of the murine placenta.

Genetic engineering of the mouse genome identified many genes that are essential for embryogenesis. Remarkably, the prevalence of concomitant placental defects in embryonic lethal mutants is highly underestimated and indicates the importance of detailed placental analysis when phenotyping new individual gene knockouts. Here we introduce high-resolution contrast-enhanced microfocus computed tomography (CE-CT) as a nondestructive, high-throughput technique to evaluate the 3D placental morphology. Using a contrast agent, zirconium-substituted Keggin polyoxometalate (Zr-POM), the soft tissue of the placenta (i.e., different layers and cell types and its vasculature) was imaged with a resolution of 3.5 µm voxel size. This approach allowed us to visualize and study early and late stages of placental development. Moreover, CE-CT provides a method to precisely quantify placental parameters (i.e., volumes, volume fraction, ratio of different placental layers, and volumes of specific cell populations) that are crucial for statistical comparison studies. The CE-CT assessment of the 3D morphology of the placentas was validated (i) by comparison with standard histological studies; (ii) by evaluating placentas from 2 different mouse strains, 129S6 and C57BL/6J mice; and (iii) by confirming the placental phenotype of mice lacking phosphoinositol 3-kinase (PI3K)-p110α. Finally, the Zr-POM-based CE-CT allowed for inspection of the vasculature structure in the entire placenta, as well as detecting placental defects in pathologies characterized by embryonic resorption and placental fusion. Taken together, Zr-POM-based CE-CT offers a quantitative 3D methodology to investigate placental development or pathologies.

Effect of the induction of transgenerational obesity on maternal-fetal parameters.

Maternal obesity can cause complications for both women and their offspring for generations. Therefore, we intended to verify the repercussions of induction of transgenerational obesity on biochemical parameters, reproductive performance, and congenital anomaly frequency in Wistar rats. Female rats were used from successive generations. The female rats of parental generation (F0, n=10) were mated to obtain their offspring (F1 generation). F1 female rats received a monosodium glutamate (MSG) solution to induce obesity (n=07) or vehicle (control, n=06) during the neonatal period. These adult female rats were classified as normal or obese using the Lee Index, mated, and delivered offspring (F2 generation), which were also evaluated for obesity using the Lee Index in adult life (F2MSG, n=13, born from obese dams) or non-obesity status (F2Control, n=12, born from control dams), and were mated in adulthood. During pregnancy, glycemia and an oral glucose tolerance test (OGTT) were analyzed. At term pregnancy, the females were sacrificed for serum biochemical profile, maternal reproductive outcomes, and fetal development. In F2MSG rats, body weight gain at early pregnancy, glycemia by OGTT, total cholesterol, high-density-lipoprotein, and alanine transaminase activity were higher compared with those of F2Control rats. F2MSG rats also presented a lower implantation number and gravid uterus weight, increased pre-implantation loss and anomaly frequency in their fetuses (F3 generation) compared with those of F2Control rats. Therefore, even without significant changes in body weight gain, obesity was established at the end of pregnancy of Wistar rats using other biomarkers. Additionally, these rats showed multiple adverse reproductive outcomes, confirming the deleterious effects that lead to obesity.

Single versus repeat doses of misoprostol for treatment of early pregnancy loss-a randomized clinical trial.

STUDY QUESTION: Does repeat administration of misoprostol for early pregnancy loss increase the treatment success rate? SUMMARY ANSWER: Repeat administration of misoprostol does not increase the treatment success rate, and is associated with more analgesics use. WHAT IS KNOWN ALREADY: Misoprostol reduces the need for surgical evacuation and shortens the time to complete expulsion in patients with early pregnancy loss. However, the impact of repeat doses of misoprostol is not clear. STUDY DESIGN, SIZE, DURATION: A randomized clinical trial was conducted in a single tertiary hospital, recruiting women with early pregnancy loss (<12 weeks), seeking medical treatment, between August 2015 and June 2016. A sample size of 160 patients was sufficient to detect a 30% decrease in treatment success. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants received 800 µg of misoprostol vaginally on Day 1, and were then randomly assigned into two groups: Patients in the single-dose group were evaluated on Day 8. Patients in the repeat-dose group were evaluated on Day 4, when they were given a repeat dose if required, and scheduled for re-evaluation on Day 8. If complete expulsion was not achieved on Day 8 (endometrial thickness >15 mm or the presence of gestational sac on transvaginal sonography), participants underwent surgical evacuation. The primary outcome was treatment success, defined as no need for surgical intervention up to Day 8. MAIN RESULTS AND THE ROLE OF CHANCE: Final analysis included 87 participants in the single-dose group and 84 participants in the repeat-dose group, out of whom 41 (48.8%) received a second dose. Treatment succeeded in 67 (77%) patients in the single-dose group and 64 (76%) patients in the repeat-dose group (RR 0.98; 95% CI 0.83-1.16; P = 0.89). Patients in the repeat-dose group reported more use of over the counter analgesics (82.1% versus 69.0%, P = 0.04). LIMITATIONS, REASONS FOR CAUTION: The study was not blinded and our definition of complete expulsion may be debated. Follow-up time was not equal in all participants, since some had a complete expulsion on Day 4 and some underwent emergent D&C before Day 8. This, however, should not affect the primary outcome. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that a single-dose protocol is superior to a repeat-dose protocol due to a comparable success rate and more favorable outcomes regarding the need for analgesic drugs. STUDY FUNDING/COMPETING INTEREST(S): We did not receive funding for this study and we declare no conflict of interest. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT02515604). TRIAL REGISTRATION DATE: 2 August 2015. DATE OF FIRST PATIENT'S ENROLMENT: 19 August 2015.

Embryo Mortality Around the Period of Maintenance of the Corpus Luteum Causes Alterations to the Ovarian Function of Lactating Dairy Cows.

Objectives were to identify cows with embryo mortality (EM) around the period of corpus luteum maintenance by interferon tau (IFNT) and to characterize ovarian function in cows that underwent EM. Lactating Holstein cows received artificial insemination (AI) (Day = 0) with semen or extender only. From Day 14 to 42 transrectal ultrasonography was performed daily to monitor ovarian dynamics and uterine contents whereas blood was collected every 48 h to determine ISG15 and MX2 mRNA abundance in blood mononuclear cells (Day 14 to 22 only) and determination of hormone concentrations. Cows were classified in the following reproductive status groups: cyclic (inseminated with extender; n = 15), pregnant (embryo present on Day 42; n = 23), no embryo (n = 23), and EM (n = 14). EM was defined as the presence of an embryo based on interferon-stimulated genes (ISG) mRNA abundance and concentrations of pregnancy-specific protein B (PSPB) above specific cutoff points but no embryo visualized by ultrasonography. Within the EM group, early EM (up to Day 22) was when ISG fold changes were above specific cutoff points from Day 18 to 22 and PSPB below 0.7 ng/ml on and after Day 24, whereas late EM (after Day 22) was when PSPB was above 0.7 ng/ml on or after Day 24 regardless of ISG expression. This experiment provided evidence that the combination of ISG expression patterns and PSPB concentrations is a reasonable method to determine EM around the period of corpus luteum maintenance by IFNT because cows with evidence of EM had patterns of ISG expression more similar to pregnant than cyclic cows or cows with no embryo. Within the EM group, only cows with late EM had delayed luteal regression and longer interovulatory intervals. No major alterations in follicular function were observed after the onset of luteolysis. Our results suggest that embryo development needs to continue beyond 22 days after AI to effectively prevent luteolysis and extend the luteal phase.

microRNAs and Endometrial Pathophysiology.

Embryo implantation requires a reciprocal interaction between the blastocyst and endometrium and is associated with complex regulatory mechanisms. Since their discovery, microRNAs became prominent candidates providing missing links for many biological pathways. In recent years, microRNAs were implicated as one of the important players in regulation of various biological and physiological endometrial related processes. This chapter aims to present recent knowledge pertaining to the diverse aspects of microRNAs in the embryo-endometrial relationship. We will focus on the role of microRNAs in decidualization and their part in natural and stimulated cycles. Next, we will present recent studies deliberating the role of microRNAs in recurrent pregnancy loss and in the important phenomenon of recurrent implantation failure. Lastly, demonstrating an important aspect of embryo implantation and invasion, we will outline few microRNA related shared pathways of implantation and carcinogenesis.

Biochemical pregnancy and spontaneous abortion in first IVF cycles are negative predictors for subsequent cycles: an over 10,000 cases cohort study.

PURPOSE: To identify whether biochemical pregnancy (BP) and spontaneous abortion (SA) cases have the same clinical characteristics in assisted reproductive therapy (ART), and to assess its predictive value for the subsequent cycles. METHODS: Retrospectively reviewed 12,174 cycles in the first in vitro fertilization and embryo transfer (IVF-ET) cycle from January 2009 to December 2012 of Peking University Third Hospital Reproductive Medical Center. Besides those patients who reached ongoing pregnancy stage, 7,598 cases were divided into three groups: group 1, lack of pregnancy (n = 6,651); group 2, BP (n = 520); and group 3, SA (n = 427). We compared the basic status of patients of the three groups, including ages, body mass index, basic hormone levels, controlled ovarian hyperstimulation protocols, amount of gonadotropin use, and endometrium thickness. The reproductive outcome of the next embryo transfer cycles of the three groups was analyzed. RESULTS: 520 patients ended as BP, and 427 patients ended as SA. The age, primary infertility proportion, body mass index, basic FSH level and basic E2 level were similar among groups. Endometrial thickness, controlled ovarian hyperstimulation protocol, Gn dosage, average oocyte retrieval and ET numbers were also similar. Multivariate analysis showed that only the age (P = 0.037, OR 1.060, 95 % CI 1.001-1.120) and endometrium thickness on hCG administration day (P = 0.029, OR 1.136, 95 % CI 1.013-1.275) may result in the differences between BP and SA groups. In the subsequent ET cycles, the total BP rate was 4.37 %, clinical pregnancy rate was 37.28 %, and miscarriage rate was 8.18 %. The clinical pregnancy rates were similar among groups. However, BP group still had the highest BP rate (P < 0.05, 7.97 vs. 4.01 % and 5.28 %), BP and SA group had higher miscarriage rate (P < 0.05, 11.76 % and 14.75 vs. 7.41 %). CONCLUSION: BP and SA in first IVF cycles had negative predictive value for subsequent ART outcomes.

Altered autonomic nervous system activity in women with unexplained recurrent pregnancy loss.

AIM: Autonomic nervous system activity was studied to evaluate the physical and mental state of women with unexplained recurrent pregnancy loss (RPL). METHODS: Heart rate variability (HRV) is a measure of beat-to-beat temporal changes in heart rate and provides indirect insight into autonomic nervous system tone and can be used to assess sympathetic and parasympathetic tone. We studied autonomic nervous system activity by measuring HRV in 100 women with unexplained RPL and 61 healthy female volunteers as controls. The degree of mental distress was assessed using the Kessler 6 (K6) scale. RESULTS: The K6 score in women with unexplained RPL was significantly higher than in control women. HRV evaluated on standard deviation of the normal-to-normal interval (SDNN) and total power was significantly lower in women with unexplained RPL compared with control women. These indices were further lower in women with unexplained RPL ≥4. On spectral analysis, high-frequency (HF) power, an index of parasympathetic nervous system activity, was significantly lower in women with unexplained RPL compared with control women, but there was no significant difference in the ratio of low-frequency (LF) power to HF power (LF/HF), an index of sympathetic nervous system activity, between the groups. CONCLUSIONS: The physical and mental state of women with unexplained RPL should be evaluated using HRV to offer mental support. Furthermore, study of HRV may elucidate the risk of cardiovascular diseases and the mechanisms underlying unexplained RPL.

Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy.

BACKGROUND: Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound. METHODS: In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology. RESULTS: The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9-11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert's membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still intact. In the subsequent resorption process first the embryo and then its membranes disappeared. CONCLUSIONS: Our results provide a temporal time course of embryo resorption. With this method, animals exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the onset of total embryo disintegration.

Inhibition of Delta-like 4 mediated signaling induces abortion in mice due to deregulation of decidual angiogenesis.

OBJECTIVE: To explore whether the Dll4/Notch1 pathway plays a key role in regulating the vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) driven decidual angiogenesis and related pregnancy through induction of a tip/stalk phenotype. METHODS: Progesterone-replaced ovariectomized pregnant mice received a single injection of YW152F (Dll4 blocking antibody, BAb) or placebo at embryonic day (E) 4.5. Animals were sacrificed at different time points; blood and uterus were collected for further analysis. Number of embryos and implantation site, uteri weight, and serum progesterone levels were assessed. Alterations in the tip/stalk phenotype were determined by quantitative immunofluorescent analysis of vascularization, Dll4 expression, cellular proliferation and apoptosis in uterine sections. RESULTS: Abrogation of Dll4 signaling leads to a promiscuous expression of Dll4, increased cell proliferation, apoptosis and vascularization at E 6.5. Such an abrogation was associated with a dramatic disruption of embryo growth and development starting at E 9.5. DISCUSSION: The observed promiscuous expression of Dll4 and the increase in cell proliferation, apoptosis and vascularization are events compatible with loss of the tip/stalk phenotype. Excessive (although very likely defective) decidual angiogenesis due to such vascular alterations is the most likely cause of subsequent interruption of embryo development and related pregnancy in Dll4 treated mice. CONCLUSIONS: Dll4 plays a key role in regulating decidual angiogenesis and related pregnancy through induction of a tip/stalk phenotype.

Long-lasting effects of neonatal bisphenol A exposure on the implantation process.

Successful implantation is the result of complex molecular interactions between the hormonally primed uterus and a mature blastocyst. This very carefully synchronized interplay of hormonal signals and feedback loops is potentially vulnerable to chemicals such as endocrine disruptors that may disrupt endocrine signaling. Bisphenol A (BPA) is one of the highest-volume chemicals produced worldwide. This chapter describes the effects of brief postnatal exposure to BPA on female reproductive performance and specifically on the uterine adaptations during the preimplantation period. We propose that an early alteration in Hoxa10 gene expression affects the functional differentiation of the preimplantation uterus as part of an altered endocrine signal transduction pathway. These molecular alterations could explain, at least in part, the adverse effects of BPA on uterine implantation. Exposure to endocrine disruptors, such as BPA, could contribute to the impaired female fertility noted over the past decades.

Comparison of reproductive outcome, including the pattern of loss, between couples with chromosomal abnormalities and those with unexplained repeated miscarriages.

AIM: Chromosomal abnormalities are an important cause of repeated miscarriage. Several studies have discussed the association between chromosomal abnormalities and repeated miscarriage. This study attempts to describe the pattern of miscarriage in this group of women and the eventual pregnancy outcome of couples with chromosomal abnormalities compared with couples with unexplained repeated pregnancy loss. MATERIAL AND METHODS: This was a retrospective study involving 795 couples with repeated miscarriages. RESULTS: Out of 795 couples, 28 (3.52%) were found to have a chromosomal abnormality (carrier group). Over half (65.5%) of the chromosomal abnormalities were balanced reciprocal translocations. After referral, this carrier group had a total of 159 pregnancies, leading to 36 live births (22.6%) among 18 couples. The after referral miscarriage rate in the chromosomal anomaly group (55.6%) was significantly (P < 0.01) higher than that in the unexplained recurrent miscarriage group (28.1%). In couples with chromosomal anomaly, the miscarriages were more likely to occur between 6 and 12 weeks' gestation. CONCLUSIONS: The encouraging cumulative live birth rate of 64.3% for couples with chromosomal anomaly and repeated miscarriage suggests that further attempts at natural conception are a viable option.

Alteration of the endometrial EGF profile as a potential mechanism connecting the alterations in the ovarian steroid hormone profile to embryonic loss in repeat breeders and high-producing cows.

Poor reproductive efficiency is a worldwide problem that has affected the dairy industry during the last several decades. In an attempt to explain the changes in reproductive physiology caused by high milk production, a model of elevated steroid metabolism in lactating dairy cows has been proposed. A slow increase in levels and low peak levels of estradiol (E2) and progesterone (P4) characterize endocrine changes in high producing cows. Similar changes have been reported in the repeat breeder cows. The abnormal changes in E2 and P4 concentrations of these cows may cause an improper uterine environment due to disturbed expression of growth factors and cytokines in the endometrium. This review focuses on the alteration in epidermal growth factor (EGF) profile in the endometrium during the estrous cycle. The normal cow has two peaks of EGF concentrations on days 2-4 and 13-14. Low concentrations of EGF on these days distinguished both high-producing and repeat breeder cows from normal cows. Alteration of the EGF profile could be found in 70 and 40% of the repeat breeder and high-producing cows, respectively. Treatment with a high dose of estradiol benzoate and an intravaginal progesterone-releasing device restored the normal EGF profile in about 70% of the affected cows. The cows having a normal EGF profile after treatment showed a higher pregnancy rate than the cows with the altered profile. Further studies to understand the etiology of the alteration in the EGF profile are needed to develop another treatment option and preventive management for this problem.

Morphology of twin and triplet equine conceptuses during weeks 3 and 4 of pregnancy.

Twin ovulations are common in horses, but twin pregnancies are rarely carried to term. Theories of how one or both twins is/are naturally eliminated in early pregnancy, termed 'embryo reduction', have been based on ultrasonographic, not morphological, studies. Here we describe conceptuses recovered transcervically between Days 15 and 28 from 31 twin and two triplet pregnancies. Signs of contact between conceptuses were deduced from those seen in one pair that remained attached by their capsules on Day 18. Signs were found on capsules in two of 10 pairs before or during fixation (immobilisation) at Days 16-17 even though contact had not been seen by ultrasound. After fixation, the signs became stronger in seven of nine unilateral pregnancies, indicated adhesion between pairs and included effects on the vitelline circulation and/or degeneration of one twin. Conceptuses recovered from five of seven unilateral twin pregnancies after the time of capsule disruption (~Day 21) evidenced embryo reduction; in the two surviving pairs, attachment between twins was near the trilaminar/bilaminar yolk-sac wall border. The findings are consistent with the notions that: (1) the capsule plays a role in initiating adhesion between twins; and (2) twin survival depends on an unencumbered trilaminar yolk-sac wall and a functional vitelline circulation.

Previous abortion is a positive predictor for ongoing pregnancy in the next cycle in women with repeated IVF failures.

Early pregnancy loss is common among women treated with assisted reproduction treatment, but whether it is a prognostic factor for success in subsequent IVF cycles is not well established. The aim of this study was to determine whether a biochemical pregnancy (BP) or spontaneous abortion (SA) affects the pregnancy rates in the following cycle. A retrospective study of 2687 women undergoing 6678 cycles between January 1998 and March 2010 was performed. Ongoing pregnancy rate (PR) per cycle was compared between patients with a pregnancy loss versus a negative ß-HCG in their previous cycles. Multivariate analysis of factors affecting ongoing pregnancy rate was performed. BP and/or SA in the first three cycles did not significantly alter the chances to conceive (16.9% patients with BP and/or SA in the previous cycle versus 16.5% patients with no previous pregnancy). From cycle 4 onwards, the presence of a previous abortion (either BP or SA) was associated with better ongoing PR (23.0% versus 11.2%, P<0.001). In conclusion, BP and/or SA in a previous cycle appears to be a positive marker for success in subsequent cycles in patients with repeated IVF failures. These results should be further investigated in this challenging group of patients.

Predictive value of serum human chorionic gonadotropin ratio, progesterone and inhibin A for expectant management of early pregnancies of unknown location.

OBJECTIVE: To evaluate serum human chorionic gonadotropin (hCG) ratio, progesterone and inhibin A as single parameters and in combination for the prediction of spontaneous resolution of pregnancies of unknown location (PUL). STUDY DESIGN: Prospective observational study of 105 consecutive patients with a diagnosis of PUL. Serum levels of hCG, progesterone and inhibin A were determined at the first visit and after 2 days. Patients were followed clinically until a final diagnosis of spontaneously resolving PUL, viable or non-viable intrauterine pregnancy, or ectopic pregnancy with need of laparoscopic intervention had been reached. Different combinations of hCG ratio (hCG at 48 h/hCG at 0 h), s-progesterone and s-inhibin A were investigated to find the best predictor for successful expectant management. RESULTS: The final pregnancy outcomes were: 52 spontaneously resolving PUL (49.5%), 37 viable intrauterine pregnancies (35.2%), 8 non-viable intrauterine pregnancies (7.6%), 7 ectopic pregnancies (6.7%), and one molar pregnancy (1.0%). An hCG ratio<0.80 predicted spontaneously resolving PUL with positive and negative predictive values (PPV and NPV), sensitivity, and specificity of 0.98, 0.78, 0.72, and 0.99, respectively. In patients with hCG ratio ≥ 0.80, a combination of s-progesterone < 20 nmol/l and s-inhibin A < 30 pg/ml predicted spontaneously resolving PUL with PPV, NPV, sensitivity and specificity of 0.92, 0.96, 0.85, and 0.98 respectively. CONCLUSION: Our results suggest that patients with PUL and hCG ratio < 0.80 display a high probability of spontaneously resolving PUL with minimum need of follow-up. In cases of hCG ratio ≥ 0.80, a combination of s-progesterone < 20 nmol/l and s-inhibin A < 30 pg/ml, may be a reliable predictor of spontaneously resolving PUL. The safety of this approach should be tested in large prospective studies.