Female-biased embryonic death from inflammation induced by genomic instability.

Genomic instability can trigger cellular responses that include checkpoint activation, senescence and inflammation1,2. Although genomic instability has been extensively studied in cell culture and cancer paradigms, little is known about its effect during embryonic development, a period of rapid cellular proliferation. Here we report that mutations in the heterohexameric minichromosome maintenance complex-the DNA replicative helicase comprising MCM2 to MCM73,4-that cause genomic instability render female mouse embryos markedly more susceptible than males to embryonic lethality. This bias was not attributable to X chromosome-inactivation defects, differential replication licensing or X versus Y chromosome size, but rather to 'maleness'-XX embryos could be rescued by transgene-mediated sex reversal or testosterone administration. The ability of exogenous or endogenous testosterone to protect embryos was related to its anti-inflammatory properties5. Ibuprofen, a non-steroidal anti-inflammatory drug, rescued female embryos that contained mutations in not only the Mcm genes but also the Fancm gene; similar to MCM mutants, Fancm mutant embryos have increased levels of genomic instability (measured as the number of cells with micronuclei) from compromised replication fork repair6. In addition, deficiency in the anti-inflammatory IL10 receptor was synthetically lethal with the Mcm4Chaos3 helicase mutant. Our experiments indicate that, during development, DNA damage associated with DNA replication induces inflammation that is preferentially lethal to female embryos, because male embryos are protected by high levels of intrinsic testosterone.

Testing the endometrium: is there enough evidence to justify clinical use?

PURPOSE OF REVIEW: Embryo implantation remains the limiting factor in assisted reproduction outcomes. To date research has mainly focused on improving embryo quality, numbers and selection as the route to improve treatment results. However, with success rates plateauing, interest in the possibility of modulating the endometrial factor is increasing, and a number of biomarkers are now available that offer the possibility of assessing endometrial function. RECENT FINDINGS: In this review, we review recent evidence for the efficacy of a number of these biomarkers, with emphasis on those that aim to enable improvement in embryo/endometrial developmental synchrony endometrium and that offer an assessment of the degree of immune activation of the endometrium. The emerging field of reproductive tract microbiome analysis is also considered. Finally, nascent biomarkers of materno-foetal dialogue, including noncoding RNAs, microvesicles and endometrial glycans are discussed. SUMMARY: Tests of potential clinical value are emerging, but further validation studies are required. The usage of innovative endometrial biomarkers provides the possibility of targeted therapies rather than the blind empirical approaches to face embryo implantation failure. It also enables the possibility of randomized controlled trials of interventions targeting the individual cause rather empirical treatments of undiagnosed recurrent implantation failure.

Nicotinamide benefits both mothers and pups in two contrasting mouse models of preeclampsia.

Preeclampsia (PE) complicates ∼5% of human pregnancies and is one of the leading causes of pregnancy-related maternal deaths. The only definitive treatment, induced delivery, invariably results in prematurity, and in severe early-onset cases may lead to fetal death. Many currently available antihypertensive drugs are teratogenic and therefore precluded from use. Nonteratogenic antihypertensives help control maternal blood pressure in PE, but results in preventing preterm delivery and correcting fetal growth restriction (FGR) that also occurs in PE have been disappointing. Here we show that dietary nicotinamide, a nonteratogenic amide of vitamin B3, improves the maternal condition, prolongs pregnancies, and prevents FGR in two contrasting mouse models of PE. The first is caused by endotheliosis due to excess levels in the mothers of a soluble form of the receptor for vascular endothelial growth factor (VEGF), which binds to and inactivates VEGF. The second is caused by genetic absence of Ankiryn-repeat-and-SOCS-box-containing-protein 4, a factor that contributes to the differentiation of trophoblast stem cells into the giant trophoblast cells necessary for embryo implantation in mice; its absence leads to impaired placental development. In both models, fetal production of ATP is impaired and FGR is observed. We show here that nicotinamide decreases blood pressure and endotheliosis in the mothers, probably by inhibiting ADP ribosyl cyclase (ADPRC), and prevents FGR, probably by normalizing fetal ATP synthesis via the nucleotide salvage pathway. Because nicotinamide benefits both dams and pups, it merits evaluation for preventing or treating PE in humans.

[The intervention effect of N-carbamoyl glutamic acid on embryo implantation disorder induced by carbon disulfide and its possible molecular mechanism].

Objective: To explore the preventive effect and possible molecular mechanism of dietary supplementation of N-carbamylglutamate (NCG) in the implantation of carbon disulfide (CS(2)) into embryo implantation disorders. Methods: embryo implantation disorder model was established by single intraperitoneal exposure to CS(2) on the 3rd, 4th, and 5th days after pregnancy. Endometrial tissues were collected for 24h after exposure to CS(2) for western-blot and immunohistochemical staining. Results: The number of embryo implantation was increased in NCG+CS(2) group, compared with CS(2) alone group. Day 4 of pregnancy when CS(2)-exposed after 24 h, the expression of pAKT protein in NCG+CS(2) group was significantly increased (P<0.05), the expression level of pAMPK protein in NCG+CS(2) group was significantly decreased, compared with CS(2) alone group, respectively. Immunohistochemical results showed that pAKT, pAMPK, AKT and AMPK proteins were expressed in luminal epithelial cells, glandular epithelial cells and stromal cells of endometrium; Day 4 of pregnancy when CS(2)-exposed after 24 h, deep staining of ATK and pAKT protein in NCG+CS(2) group, the AMPK and pAMPK protein staining became lighter. Conclusion: Dietary supplementation of NCG can interfere with the embryo loss induced by CS(2) by altering the total amount of AKT/AMPK molecules.

Macrophage depletion and TNF-α inhibition prevent resorption in CBA/J × DBA/2 model of CpG-induced abortion.

OBJECTIVE: To elucidate the mechanism by which embryo-resorption was enhanced by pathogenic CpG ODN motif in abortion-prone CBA/J × DBA/2 model and to develop a counter strategy for normal pregnancy outcome. METHODS: This is an animal model-based study. Abortion-prone model is established by CBA/J × DBA/2. An infection was mimicked by CpG ODN injection. RESULTS: Embryo-resorption was readily induced by CpG ODN in low doses of CpG ODN (∼25 µg/dam) when intraperitoneally (IP) injected on gestational day(gd) 6.5 in male DBA/2 mated CBA/J female mice. A more modest decline in Progesterone(P4), but not Estrogen(E2) was observed after exposure to CpG ODN in the model. P4 supplement fail to improve pregnancy outcomes, even at pharmocology dose. CpG ODN-induced fetal resorption is prevented by the treatment of anti-F4/80 or by that of anti-TNFα.In the implantation sites, the treatment of anti-F4/80 inhibits the increase both of F4/80(+) macrophage proportion and TNF-αexpression level which are induced by CpG ODN. The anti-TNFαtreatment also recovers CpG ODN-induced reduction of CD4(+)Foxp3(+) T cells. CONCLUSION: Circulating P4 is not responsible for the process by which CpG ODN-induced embryonic resorption in an abortion-prone mice. Macrophage depletion and TNF-α inhibition are really noteworthy for CpG ODN-induced pregnancy disruption.

Pregnancy losses in cattle: potential for improvement.

For heifers, beef and moderate-yielding dairy cows, it appears that the fertilisation rate generally lies between 90% and 100%. For high-producing dairy cows, there is a less substantive body of literature, but it would appear that the fertilisation rate is somewhat lower and possibly more variable. In cattle, the major component of embryo loss occurs in the first 16 days following breeding (Day 0), with emerging evidence of greater losses before Day 8 in high-producing dairy cows. In cattle, late embryo mortality causes serious economic losses because it is often recognised too late to rebreed females. Systemic concentrations of progesterone during both the cycle preceding and following insemination affect embryo survival, with evidence of either excessive or insufficient concentrations being negatively associated with survival rate. The application of direct progesterone supplementation or treatments to increase endogenous output of progesterone to increase embryo survival cannot be recommended at this time. Energy balance and dry matter intake during the first 4 weeks after calving are critically important in determining pregnancies per AI when cows are inseminated at 70-100 days after calving. Level of concentrate supplementation of cows at pasture during the breeding period has minimal effects on conception rates, although sudden reductions in dietary intake should be avoided. For all systems of milk production, more balanced breeding strategies with greater emphasis on fertility and feed intake and/or energy must be developed. There is genetic variability within the Holstein breed for fertility traits, which can be exploited. Genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilisation and the establishment of pregnancy, but also, in the future, could identify genes responsible for improved embryo survival. Such information could be incorporated into breeding objectives in order to increase the rate of genetic progress for embryo survival. In addition, there is a range of easily adoptable management factors, under producer control, that can either directly increase embryo survival or ameliorate the consequences of low embryo survival rates. The correction of minor deficits in several areas can have a substantial cumulative positive effect on herd reproductive performance.

Peri-ovulatory putrescine supplementation reduces embryo resorption in older mice.

STUDY QUESTION: Does peri-ovulatory putrescine supplementation of older mice improve oocyte quality and reduce the incidence of embryo resorption? SUMMARY ANSWER: Peri-ovulatory putrescine supplementation in older mice improved oocyte quality, as indicated by increased blastocyst cell numbers and reduced the incidence of embryo resorption. WHAT IS KNOWN ALREADY: Rodents exhibit a transient rise of ornithine decarboxylase (ODC) and putrescine in the ovaries during ovulation. Older mice exhibit reduced ovarian ODC activity during ovulation. Supplementation of in vitro maturation medium with putrescine reduces oocyte aneuploidy rates of older mice. STUDY DESIGN, SIZE, DURATION: The rationale was to correct ovarian putrescine deficiency in older mice by peri-ovulatory putrescine supplementation in drinking water and to observe the reproductive consequences of this intervention. This project was conducted between 2010 and 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS: Older mice (9-11 months of age) were given regular drinking water (control) or drinking water with 1% putrescine dihydrochloride (62 mM) for 2-4 days before mating. Plugged mice were then withdrawn from putrescine supplementation. Blastocysts were retrieved on 3.5 days post coitum (dpc) for the determination of cell numbers. For resorption analyses, mice were killed on 9.5 dp or 12.5 dpc, and implantation sites were dissected to determine the embryo status. For birth studies, mice were examined every morning between 16.5 and 23.5 dpc. Births were recorded as live or stillbirth. MAIN RESULTS AND THE ROLE OF CHANCE: We demonstrated that deficiency of ovarian putrescine in older mice can be restored by peri-ovulatory putrescine supplementation in drinking water. Putrescine supplementation in older mice increased blastocyst cell numbers (from 40 to 54; P < 0.0001, t-test), reduced embryo resorption rates (from 41.1 to 15.4% in old C57BL/6 mice, P < 0.0001, Fisher's exact test; from 14.2 to 6.4% in old CF1 mice, P = 0.004, Fisher's exact test), and doubled the number of live born pups. Furthermore, exogenous putrescine exhibited rapid absorption and excretion, and showed no toxicity to mothers or fetuses. LIMITATIONS, REASONS FOR CAUTION: The mechanism of putrescine action in oocytes and/or ovaries remains unclear. WIDER IMPLICATIONS OF THE FINDINGS: Peri-ovulatory putrescine deficiency in older mice appears to adversely impact on oocyte maturation resulting in poor quality embryos (as assessed by blastocyst cell numbers) and early embryo death. This study demonstrates a natural and simple remedy to improve oocyte quality in older women. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the NSERC, the March of Dimes Foundation, and the National Natural Science Foundation of China. The authors declare no competing interest.

Anakinra and etanercept prevent embryo loss in pregnant nonobese diabetic mice.

Bacteria and viruses activate the host innate immune response via Toll-like receptor (TLR)-involved signaling and potentially cause pregnancy failure. TLR7 and TLR9 respond to single-stranded RNA (a viral intermediate) and hypomethylated CpG DNA motifs (specific molecular constituents of bacteria) respectively. In this study, we treated murine RAW264.7 cells with R837, CpG1826, or a combination of the two. RT-PCR was performed to detect cytokines, Tlr7, and Tlr9. WT and nonobese diabetic murine embryo resorption models were established by i.p. injections of TLR7 and TLR9 ligands. Neutralizing antibodies and the IL1ß and TNFα inhibitors were used. The specific inhibitors anakinra and etanercept effectively prevented TLR7 and TLR9 ligand-induced embryo loss. Notably, this effect was not observed in decidual NK cell-depleted mice. Our findings suggest that anakinra and etanercept may have potential for preventing TLR7 or TLR9 ligand-induced abortion in the presence of decidual NK cells.

Implantation of fresh and thawed-warmed embryos in single embryo transfer cycles: interpreting the initial beta-HCG.

Little is known about the effects of human embryo cryopreservation on developmental potential. Initial beta-HCG, indicating embryo implantation, was measured in 322 single embryo transfer cycles (246 fresh and 76 thawed-warmed). Median initial beta-HCG was higher for fresh compared with thawed-warmed transfers (126 versus 100 mIU/ml; P = 0.04). Blastocyst slow cooling resulted in a lower initial beta-HCG compared with vitrification (P = 0.01). Live birth rates were lower for blastocyst slow cooling (25%) compared with vitrification (71%) and fresh transfer (70%). We conclude that cryopreservation may impair an embryo's ability to produce beta-HCG, but that vitrification does not impair developmental potential.

NKG2D blockade inhibits poly(I:C)-triggered fetal loss in wild type but not in IL-10-/- mice.

Infection and inflammation can disturb immune tolerance at the maternal-fetal interface, resulting in adverse pregnancy outcomes. However, the underlying mechanisms for detrimental immune responses remain ill defined. In this study, we provide evidence for immune programming of fetal loss in response to polyinosinic:polycytidylic acid (polyI:C), a viral mimic and an inducer of inflammatory milieu. IL-10 and uterine NK (uNK) cells expressing the activating receptor NKG2D play a critical role in poly(I:C)-induced fetal demise. In wild type (WT) mice, poly(I:C) treatment induced expansion of NKG2D(+) uNK cells and expression of Rae-1 (an NKG2D ligand) on uterine macrophages and led to fetal resorption. In IL-10(-/-) mice, NKG2D(-) T cells instead became the source of fetal resorption during the same gestation period. Interestingly, both uterine NK and T cells produced TNF-α as the key cytotoxic factor contributing to fetal loss. Treatment of WT mice with poly(I:C) resulted in excessive trophoblast migration into the decidua and increased TUNEL-positive signal. IL-10(-/-) mice supplemented with recombinant IL-10 induced fetal loss through NKG2D(+) uNK cells, similar to the response in WT mice. Blockade of NKG2D in poly(I:C)-treated WT mice led to normal pregnancy outcome. Thus, we demonstrate that pregnancy-disrupting inflammatory events mimicked by poly(I:C) are regulated by IL-10 and depend on the effector function of uterine NKG2D(+) NK cells in WT mice and NKG2D(-) T cells in IL-10 null mice.

Management Strategies Aiming to Improve Horse Welfare Reduce Embryonic Death Rates in Mares.

The objective of this retrospective study was to evaluate the effect of management strategies aiming to improve animal well-being on pregnancy and embryonic death (ED) rates. Breeding records of a cohort of 1206 Thoroughbred mares brought to a stallion station facility, to be bred with the stallions housed there, were evaluated during ten breeding seasons. Mares were blocked according to management strategies in two groups: Stress and Relax. Strategies used to improve animal well-being (Relax group) were as follows: stopping the teasing routine, reducing or eliminating stall confinement, reducing the number of mares per group and maintaining herd stability during the breeding season. In barren mares, the pregnancy rate was higher in the Relax group (91.8%) when compared to the observed in Stress group (84.7%). However, no difference in pregnancy rates were observed (Stress = 85.2% vs. Relax = 86.2) in foaling mares. ED rate was higher in barren and foaling mares of the Stress group mares (25.5% and 26.8%, respectively) compared with the Relax group (16.1% and 14.7%, respectively). No significant differences were observed on foal heat pregnancy rate between groups; yet, the embryo loss on foal heat was significant reduced in Relax mares (Relax = 8.7% vs Stress = 24.5%). In conclusion, management strategies aimed to reduce social stress can reduce early pregnancy losses and the average cycles per pregnancy, improving reproductive performance in mares.

Rationalizing the management of pregnancies of unknown location: temporal and external validation of a risk prediction model on 1962 pregnancies.

STUDY QUESTION: Can we accurately define a group of pregnancies of unknown location (PULs) as low risk in order to safely reduce follow-up for these pregnancies and allocate resources to pregnancies at an increased risk of being ectopic? SUMMARY ANSWER: Prediction model M4 classified around 70% of PULs as low risk, of which around 97% were later characterized as failed PULs or intrauterine pregnancies (IUPs), while still classifying 88% of ectopic pregnancies as high risk. WHAT IS KNOWN ALREADY: Depending on the level of suspicion of ectopic pregnancy (EP), women with a PUL receive a lengthy follow-up in order to confirm the location and viability of the pregnancy. STUDY DESIGN, SIZE, DURATION: A multi-centre diagnostic accuracy study of 1962 patients was carried out between 2003 and 2007 for retrospective temporal validation and between 2009 and 2011 for prospective external validation. The reference standard is the final characterization of PUL as failed pregnancies or IUPs (low risk), or as ectopic pregnancies (high risk). M4 is a multinomial logistic regression model based on the serum human chorionic gonadotrophin (hCG) levels at presentation and 48 h later. PARTICIPANTS/MATERIALS, SETTING, METHODS: Temporal validation data from 1341 PULs collected at St George's Hospital in London were available, of which 53% were failed, 39% were intrauterine and 8% were ectopic pregnancies. External validation data from 621 PULs collected at four other London-based teaching hospitals were available, of which 63% were failed, 22% were intrauterine and 15% were ectopic pregnancies. MAIN RESULTS AND THE ROLE OF CHANCE: The EP rate varied between 8 and 16% across the five hospitals. At St George's, 980 [73.1%, 95% confidence interval (CI): 70.5-75.4] PULs were considered low risk. Of these, 963 were failed PULs or IUPs (98.3%, 95% CI: 97.2-98.9) and 17 were ectopic pregnancies. At the other four hospitals, 62-75% were considered low risk, with 96-98% of these turning out to be failed PUL or IUP. Eighty-five percent (95% CI: 76.8-90.2) of the ectopic pregnancies were considered high risk at St George's, compared with 80-92% in the other hospitals. LIMITATIONS, REASONS FOR CAUTION: Of total, 120 patients had been excluded due to loss to follow-up, and a further 102 patients because of missing hCG levels due to differences in local clinical practice. There are variations in the definition of a PUL used in different countries. WIDER IMPLICATIONS OF THE FINDINGS: The suggested protocol could safely reduce the follow-up in the majority of PUL such that units could increase the focus on women at a risk of complications. This would lead to a change in the management of the majority of women with a PUL and a more efficient use of resources. At the end of the manuscript, we provide a link to enable clinicians to use the protocol. STUDY FUNDING/COMPETING INTEREST(S): B.V.C. is supported by a postdoctoral fellowship from the Research Foundation Flanders (FWO). K.V.H. is supported by a fellowship from the Flanders' Agency for Innovation by Science and Technology (IWT-Vlaanderen), by the Research Council KU Leuven (GOA MaNet), by the Flemish Government (iMinds) and by the Belgian Federal Science Policy Office (IUAP P7/DYSCO). T.B. is supported by the Imperial Healthcare NHS Trust NIHR Biomedical Research Centre. No competing interests are declared.

Effect of feeding level on luteal function and progesterone concentration in the vena cava during early pregnancy in gilts.

This study assessed the effect of feeding level on progesterone concentration in the caudal vena cava during early pregnancy in gilts. Twenty-four Landrace gilts were allocated to either a high (2.8±0.02) or a low (1.5±0.01 kg day⁻¹) feeding level at Day 0 of pregnancy. Serial blood samples were collected every 15 min for 3 h before and 3 h after feeding on Days 6 and 9 of pregnancy. Embryo survival and development as well as in vitro luteal progesterone production were assessed at Day 10 of pregnancy. Progesterone concentration in the vena cava was pulsatile with gilts on the high feeding level having more pulses compared with Low gilts on Day 9 of pregnancy (P<0.05). On Day 6 the number of pulses did not differ significantly between treatments; however, the average progesterone concentration in the vena cava tended to be higher in the gilts on the high feeding level (P<0.10). Embryo survival at Day 10 was 92±3% for High gilts compared with 77±3% for Low gilts (P<0.05). No difference in embryo development between the treatments was seen. There was no difference between treatments in in vitro secretion of progesterone by luteal tissue. In conclusion, a high plane of nutrition positively affects progesterone secretion by the ovaries in early pregnancy.

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.

Intradermal sex hormone desensitization for relief of premenstrual symptoms may improve the obstetric outcome of women with recurrent pregnancy loss.

The aim of this study was to determine whether women with recurrent pregnancy loss (RPL) and concurrent premenstrual syndrome (PMS) who underwent desensitization with sex hormones had an improved obstetric outcome. This manuscript summarizes a 10 year open label prospective follow up study of 26 women with RPL, aged 25-42 with 3-8 previous miscarriages and PMS, who had hormone hypersensitivity on skin testing. Skin testing was positive to estradiol in 23 women, progesterone in 20 women and to both estrogen and progesterone in 17 women. Amelioration of the symptoms of PMS (according to the VAS) was seen in 21 of 26 patients after desensitization with small doses of sex hormones intradermally. There was long term and stable reduction of severe PMS in 21 of 26 patients after desensitization. Five women conceived after skin testing, prior to desensitization. Sixteen of 26 women (61%) had subsequent live births. Five women had two subsequent live births in the subsequent pregnancy. There were no obstetric complications. Five women had two subsequent pregnancies with live births. It seems that correction of sex hormone hypersensitivity was accompanied by relief of persistent PMS, may have a positive effect on the chances of a successful pregnancy.

Single curettage endometrial biopsy injury in the proliferative phase improves reproductive outcome of subsequent in vitro fertilization-embryo transfer cycle in infertile patients with repeated embryo implantation failure.

PURPOSE OF INVESTIGATION: To evaluate the effectiveness of single curettage endometrial biopsy injury (EBI) in the proliferative phase for in vitro fertilization-embryo transfer (IVF-ET) outcome of the subsequent cycle in infertile patients with repeated embryo implantation failure (EIF). MATERIALS AND METHODS: Of 89 patients who repeated EIF three times following transfer of morphologically good embryos and/or blastocysts, 40 patients chose curettage EBI prior to the subsequent IVF-ET cycle. Using a three-mm wide curette, EBI was performed once between days 6 and 12 of the spontaneous cycle. Their IVF-ET outcomes in the subsequent cycle were compared with those in 49 patients who did not opt for EBI. RESULTS: The clinical pregnancy rate (37.5% vs 12.2%), embryo implantation rate (23.6% vs 6.3%), and ongoing pregnancy rate (25.0% vs 8.2%) were significantly higher in the EBI group than in the non-EBI group. No serious complaints and complications were noted. CONCLUSION: Single curettage EBI in the proliferative phase of the preceding cycle significantly improved IVF-ET outcome in infertile patients with repeated EIF.

Hollow fiber vitrification provides a novel method for cryopreserving in vitro maturation/fertilization-derived porcine embryos.

In vitro matured (IVM) oocytes have been used to create genetically modified pigs for various biomedical purposes. However, porcine embryos derived from IVM oocytes are very cryosensitive. Developing improved cryopreservation methods would facilitate the production of genetically modified pigs and also accelerate the conservation of genetic resources. We recently developed a novel hollow fiber vitrification (HFV) method; the present study was initiated to determine whether this new method permits the cryopreservation of IVM oocyte-derived porcine embryos. Embryos were created from the in vitro fertilization of IVM oocytes with frozen-thawed sperm derived from a transgenic pig carrying a humanized Kusabira-Orange (huKO) gene. Morula-stage embryos were assigned to vitrification and nonvitrification groups to compare their in vitro and in vivo developmental abilities. Vitrified morulae developed to the blastocyst stage at a rate similar to that of nonvitrified embryos (66/85, 77.6% vs. 67/84, 79.8%). Eighty-eight blastocysts that developed from vitrified morulae were transferred into the uteri of three recipient gilts. All three became pregnant and produced a total of 17 piglets (19.3%). This piglet production was slightly lower, albeit not significantly, than that of the nonvitrification group (27/88, 30.7%). Approximately half of the piglets in the vitrification (10/17, 58.8%) and nonvitrification (15/27, 55.6%) groups were transgenic. There was no significant difference in the growth rates among the piglets in the two groups. These results indicate that the HFV method is an extremely effective method for preserving cryosensitive embryos such as porcine in vitro maturation/fertilization-derived morulae.

Endometrial injury to overcome recurrent embryo implantation failure: a systematic review and meta-analysis.

Mechanical endometrial injury (biopsy/scratch or hysteroscopy) in the cycle preceding ovarian stimulation for IVF has been proposed to improve implantation in women with unexplained recurrent implantation failure (RIF). This is a systematic review and meta-analysis of studies comparing the efficacy of endometrial injury versus no intervention in women with RIF undergoing IVF. All controlled studies of endometrial biopsy/scratch or hysteroscopy performed in the cycle preceding ovarian stimulation were included and the primary outcome measure was clinical pregnancy rate. Pooling of seven controlled studies (four randomized and three non-randomized), with 2062 participants, showed that local endometrial injury induced in the cycle preceding ovarian stimulation is 70% more likely to result in a clinical pregnancy as opposed to no intervention. There was no statistically significant heterogeneity in the methods used, clinical pregnancy rates being twice as high with biopsy/scratch (RR 2.32, 95% CI 1.72-3.13) as opposed to hysteroscopy (RR 1.51, 95% CI 1.30-1.75). The evidence is strongly in favour of inducing local endometrial injury in the preceding cycle of ovarian stimulation to improve pregnancy outcomes in women with unexplained RIF. However, large randomized studies are required before iatrogenic induction of local endometrial injury can be warranted in routine clinical practice. Some women undergoing IVF treatment fail to conceive despite several attempts with good-quality embryos and no identifiable reason. We call this 'recurrent implantation failure' (RIF) where the embryo fails to embed or implant within the lining of the womb. Studies have shown that inducing injury to the lining of the womb in the cycle before starting ovarian stimulation for IVF can help improve the chances of achieving pregnancy. Injury can be induced by either scratching the lining of the womb using a biopsy tube or by telescopic investigation of the womb using a camera. We performed a collective review of the available good-quality studies that used the above two methods in the cycle prior to starting ovarian stimulation for IVF. We pooled results from seven studies, which included 2062 women with RIF and assessed the difference in clinical pregnancy rates for those undergoing injury to the womb lining compared with no injury prior to IVF. The results suggest that inducing injury is 70% more likely to result in a clinical pregnancy as opposed to no treatment. Furthermore, scratching of the lining was 2-times more likely to result in a clinical pregnancy compared with telescopic evaluation of the lining of the womb. This study suggests that in women with RIF, inducing local injury to the womb lining in the cycle prior to starting ovarian stimulation for IVF can improve pregnancy outcomes. However, large studies are required before this can be warranted in routine clinical practice.

Temporal candidate gene expression in the sow placenta and embryo during early gestation and effect of maternal Progenos supplementation on embryonic and placental development.

The present study characterised gene expression associated with embryonic muscle development and placental vascularisation during early gestation in the pig and examined effects of Progenos supplementation in early pregnancy. Tissues were collected from commercial multiparous sows (n = 48) from Days 16 to 49 of gestation. In the placenta, qPCR revealed that vascular endothelial growth factor (VEGFA) expression did not change from Day 17 to 49 of gestation; however, KDR receptor and angiopoietin-1 and -2 expression were differentially regulated, with periods of high expression corresponding to two critical phases of angiogenesis in the pig. In the embryo, the pattern of myogenesis-related gene expression was consistent with available literature. A commercially available nutritional supplement Progenos (20 g day⁻¹ L-arginine) added to the diet of sows from either Day 15 to 29 (P15-29; n = 33), Day 30 to 44 (n = 29) or from Day 15 to 44 (n = 76) of gestation tended to increase (P = 0.058) embryonic growth rate compared with non-supplemented controls (n = 79) and angiogenin expression was higher (P = 0.028) at Day 30 of gestation in placentae from sows on the P15-29 Progenos treatment. These results are consistent with proposed beneficial effects of l-arginine on early embryonic development and placental vascularisation.

Recurrent pregnancy loss - beyond evidence based medicine.

Patients with five or more unexplained recurrent pregnancy losses (RPL) have a poor prognosis for a subsequent delivery compared to patients with two to three RPL. Treatment guidelines are needed to address this select group of patients.