A Short-Term Altrenogest Treatment Post-weaning Followed by Superovulation Reduces Pregnancy Rates and Embryo Production Efficiency in Multiparous Sows

Although embryo transfer (ET) is a biotechnology ready for the swine industry, there are factors to be solved, the availability of embryo donors as one. Multiparous sows as donors ought to be considered since weaning is a natural and efficient method for estrus synchronization. In addition, superovulation treatments at weaning are effective in increasing the efficiency of donor embryo production. However, ET programs typically require more donors than those available from a single weaning, imposing grouping several weanings to establish a batch for ET. Since short-term administration of Altrenogest is effective in delaying estrus after weaning without effects on ovulation and embryo development, we investigated how Altrenogest combined with superovulation would affect reproductive parameters and embryo quality and quantity of weaned multiparous donor sows. The sows were administered Altrenogest from the day of weaning for 14 (SS-14 group; N = 26), 7 (SS-7 group; N = 31) and 4 (SS-4 group; N = 32) days. The sows were superovulated with eCG 24 h after the last administration of Altrenogest and with hCG at the onset of estrus. Sows not treated with Altrenogest that were superovulated with eCG 24 h post-weaning and hCG at the onset of estrus (SC group; N = 37) and sows with natural estrus after weaning (C group; N = 34) were used as control groups. The percentage of sows showing estrus within 10 days was not affected by the treatment, but the interval from Altrenogest withdrawal to estrus was longer (P < 0.05) in the SS groups than the interval from weaning to estrus in the controls. SS treatments increased (P < 0.05) the percentage of sows with ovarian cysts and the development of polycystic ovaries. The pregnancy and the fertilization rates, and the overall embryo production efficiency were also negatively affected by the SS treatments (P < 0.05). Interestingly, almost 70% of the structures classified as unfertilized oocytes or degenerated embryos in sows from the SS groups were immature oocytes. In conclusion, although superovulation of weaned sows was highly efficient, short-term administration of Altrenogest in combination with superovulation had negative effects on most of the reproductive parameters assessed, particularly affecting the overall efficiency of pregnancy and embryo production.

The incidence of hypertensive disorders of pregnancy following sperm donation in IVF: an Australian state-wide retrospective cohort study

STUDY QUESTION Does IVF using donor sperm increase the risk of hypertensive disorders of pregnancy and fetal growth restriction (FGR)? SUMMARY ANSWER IVF conceptions arising from sperm donation are not associated with an increased risk of hypertensive disorders of pregnancy or FGR. WHAT IS KNOWN ALREADY It has been hypothesized that the absence of prior exposure to factors within the paternal ejaculate increases the risk of preeclampsia and FGR among nulliparous women or women with a new partner—the concept of ‘primipaternity’. It remains unclear which element of the ejaculate is responsible: the sperm cell or the constituents of seminal fluid. IVF pregnancies arising from donor sperm where the seminal fluid is absent provide a unique opportunity to test the theory of primipaternity and the relative contribution of the sperm cell. Pregnancies conceived via artificial reproductive technology are at increased risk of preeclampsia and FGR. STUDY DESIGN, SIZE, DURATION Theories about the development of preeclampsia and the relative contribution of spermatic factors were explored by comparing the risk of hypertensive disorders of pregnancy and FGR among IVF pregnancies conceived with autologous gametes (own eggs and partner sperm) and those conceived with donor sperm, donor egg (and partner sperm) and donor embryo. To do this, we performed a retrospective cohort analysis of pregnancy outcomes among singleton pregnancies (n = 15 443) conceived through fertility clinics within Australia between 2009 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS All pregnancies resulting in a singleton pregnancy delivering after 20 weeks’ gestation were included. The cohort was divided into donor sperm, donor egg and donor embryo (where both gametes came from a donor to create an embryo, or in a surrogate pregnancy) groups. We also compared the data with a control group, defined as IVF-conceived pregnancies from autologous cycles. A multivariable regression model was used to calculate an adjusted odds ratio (aOR). MAIN RESULTS AND THE ROLE OF CHANCE The final cohort contained 1435, 578 and 239 pregnancies conceived by donor sperm, donor egg and donor embryo, respectively, and 13 191 controls. There were a very small number of women lost to follow-up (31 women; 0.2% of total cohort). Compared to control pregnancies, there was no increase in the risk of hypertensive disorders among pregnancies conceived via donor sperm (aOR 0.94; 95% CI 0.73–1.21). Subgroup analysis was performed for a cohort where parity was known (n = 4551), and of these, 305 multigravida pregnancies were conceived via donor sperm. Among this cohort, no increased risk of preeclampsia or pregnancy-induced hypertension was found (aOR 1.18; 95% CI: 0.69–2.04) as a result of primipaternity (new sperm donor). A significantly increased risk for hypertensive disorders of pregnancy was associated with the use of donor eggs (but partner sperm; aOR 2.34; 95% CI 1.69–3.21). However, the association was no greater among pregnancies conceived with donor embryos (i.e. donated egg and sperm; aOR 2.0; 95% CI 1.25–3.17) than among the donor oocyte group. The overall incidence of FGR (defined as birthweight <10th centile) was 18%. There were no significant differences observed between donor sperm, or donor embryo pregnancies; however, egg donation was associated with a 1.5-fold increase in FGR. LIMITATIONS, REASONS FOR CAUTION This study was limited by a lower than expected rate of hypertensive disorders of pregnancy (n = 862, 5.6%), which is contrary to the well-established increased risk among women using IVF. However, this is likely to be evenly distributed across the study groups and, therefore, unlikely to have introduced significant bias. WIDER IMPLICATIONS OF THE FINDINGS These findings suggest that exposure to new sperm may not be implicated in the pathogenesis of preeclampsia. The mechanism of increased risk seen in conceptions arising from egg or embryo donation remains unclear. Further investigation is required to elucidate these mechanisms and, ultimately, improve pregnancy outcomes following IVF. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Australian Commonwealth Government—Graduate Research Scheme (A.K.). Salary support was provided by the National Health and Medical Research Council of Australia (S.T.), Mercy Foundation (A.L.), and the Department of Obstetrics and Gynaecology at the University of Melbourne (R.H.). There are no competing interests.

An Unusual Presentation of Partial Hemolysis, Elevated Liver Enzyme Levels, and Low Platelet Levels Syndrome in a Case of in vitro Fertilization-DEM Pregnancy

ABSTRACT Pregnancy in women with systemic lupus erythematosus (SLE) or autoimmune conditions is complicated by complementmediated injury and is associated with increased risk of preeclampsia. Immunological intolerance between the mother and the fetus may play an important role in the pathogenesis of preeclampsia. The present case highlights the influence of susceptibility to preeclampsia/hemolysis, elevated liver enzyme levels, and low platelet levels (HELLP) syndrome due to preexisting autoimmune conditions. In vitro fertilization (IVF)- Donor embryo pregnancy (DEM) pregnancy is one such unique scenario where there exists partial immunological disparity between the native intrauterine environment and the implanted donor embryo that could predispose to autoimmune problems in pregnancy. This case illustrates a similar scenario in which a pregnant woman with IVF-DEM conception and antinuclear antibody (ANA) positive status manifested with a normotensive partial HELLP syndrome which was reiterated by the fact that there was clinical evidence of retroperitoneal hemorrhage intraoperatively during cesarean along with the neonate who was diagnosed to have L-carnitine deficiency, which is unique to fetuses with mothers of HELLP syndrome. How to cite this article Garg N, Govind R, Avula S, Rao KA. An Unusual Presentation of Partial Hemolysis, Elevated Liver Enzyme Levels, and Low Platelet Levels Syndrome in a Case of in vitro Fertilization-DEM Pregnancy. Int J Infertil Fetal Med 2016;7(2):73-75.

84 PAIRS OF BLASTOMERES FROM BOVINE DAY 5 MORULAE ARE ABLE TO CONTRIBUTE TO INNER CELL MASS AND TROPHECTODERM IN CHIMERIC EMBRYOS GENERATED BY AGGREGATION WITH TWO DAY 4 MORULAE

The multiplication of high-value embryos by chimera formation using asynchronic aggregation is a promising alternative to embryonic cell nuclear transfer. Single blastomeres from a donor embryo are aggregated with 2 host embryos, thus several chimeras can be constructed per donor embryo. Due to the advanced developmental stage, the donor blastomeres are likely to contribute to the inner cell mass (ICM) and later give rise to the embryo proper, whereas the host embryos form extra-embryonic tissues. To test if pairs of blastomeres from Day 5 morulae are able to form the ICM when aggregated with 2 Day 4 host embryos, we produced transgenic donor embryos carrying a fluorescent reporter gene (enhanced green fluorescent protein, eGFP) by using semen from an eGFP transgenic bull (Reichenbach et al. 2010 Transgenic Res. 19, 549–556) for in vitro fertilization and in vitro host embryos produced by a standard procedure. The zona pellucida of all embryos was removed by treatment with 1 mg mL–1 pronase. Donor embryos were assessed for eGFP expression by fluorescence microscopy and disaggregated by gentle pipetting after incubation in Mg2+- and Ca2+-free medium. Pairs of blastomeres were then placed between 2 host embryos and cultured individually in a well-of-the-well culture dish. On Day 6 after aggregation, fully developed blastocysts were assessed for eGFP fluorescence. In 3 replicates, n = 30 chimeras were produced by aggregation; 13 (43%) developed to blastocysts, of which 2 (15%) showed local eGFP expression in the ICM and 7 (54%) showed a generalized expression. From the results of this study we conclude that Day 5 morulae may be multiplied in an efficient manner by using the chimera formation technique, which makes this approach applicable to ex vivo-derived embryos. In future investigations we will study the effect of using donor blastomeres from either the inside or outside of the donor morula and test the use of tetraploid host embryos to increase the rate of blastocysts with the desired genotype in the ICM. Finally, we aim to introduce this multiplication approach to the production of genotyped embryos with a genomic estimated breeding value (gEBV) and intend to produce calves with identical gEBV.Funded by the Bavarian Research Foundation (AZ-1031–1).

146 BOVINE EMBRYO AGGREGATION TO MULTIPLY ELITE GENOTYPES

Each in vitro produced bovine embryo has only a limited chance of developing into a live calf. As genetically superior embryos can now be identified (Humblot et al. 2010 Vet. Med. Int.), maximizing these odds is desirable. We aim to multiply the elite genetics of selected male embryos by aggregating them with female embryos and generating germ line chimaeras. We postulate that this approach will result in functionally male cattle that produce sperm derived from the embryo of high genetic value. In this study, we evaluated the number and developmental stage of male cleavage-stage blastomeres required for efficient sex conversion and chimaerism. On Day 4, at the 12- to 16-cell stage, each presumptive male embryo donor (generated from Y-chromosome-sorted sperm) was dissociated into 1/4 (3–4 cells), 1/2 (6–8 cells) or 3/4 (9–12 cells) portions. One-quarter of each donor embryo was kept for future genotyping. Each donor portion was aggregated with a whole Day 4 presumptive female embryo generated from X-chromosome-sorted sperm from a different sire. On Day 7, blastocysts were assessed for blastomere incorporation and suitable quality embryos transferred individually to recipients. Development of individual aggregates to suitable blastocysts was not significantly different between 1/4-, 1/2-, and 3/4-aggregates or control whole male embryos (11/49 = 22%, 20/49 = 41% 15/50 = 30%, and 11/37 = 30%, respectively). However, aggregating three 1/4 embryos resulted in a 2.3-fold increase in the number of blastocysts produced per donor embryo compared to controls (11/16 = 69% v. 11/37 = 30%). Following embryo transfer of different aggregate groups (1/4, 1/2, and 3/4) and controls, there was neither a significant difference for pregnancy establishment on Day 35 (5/11 = 45%, 9/15 = 60%, 8/13 = 62%, and 5/10 = 50%, respectively), nor on Day 150 of gestation (2/11 = 18%, 3/15 = 20%, 4/13 = 31%, and 4/10 = 40%, respectively). Most of the aggregate fetuses recovered around Day 150 were phenotypically male (1/4 = 2 of 2, 1/2 = 1 of 3, and 3/4 = 4 of 4 fetuses; 7/9 = 78% overall). Expression of the female-specific mRNA for XIST (X-inactive specific transcript) was detected in liver and/or heart from aggregate male fetuses (3 from 3/4 aggregates and 1 from a 1/4 aggregate; 4/7 = 57% overall), indicating somatic cell sex chimaerism. Using sex-specific sequences of the amelogenin gene (AMELX and AMELY), no Y-chromosomal contribution was detected in liver or heart of the two phenotypically female aggregate fetuses. A recent asynchronous aggregation experiment, whereby three 1/4 male Day 5 embryos were each aggregated with a single Day 4 female embryo, resulted in a 4.6-fold increase in the number of blastocysts produced per donor embryo compared to controls (39/17 = 229% v. 22/44 = 50%). In summary, embryo aggregation more than doubled the number of blastocysts that potentially contained the desired genetics of a donor embryo. The majority of the resulting fetuses were male and developed to Day 150 of gestation at rates comparable to controls. Future studies will determine the minimum number of male blastomeres required to ensure sex conversion and colonisation of the germ line to reliably generate bull calves. Supported by MSI C10X1002.

176PRODUCTION OF TRANSFERABLE EMBRYOS IN BRAHMAN COWS TREATED WITH BOVINE SOMATOTROPIN

It is the goal of numerous research laboratories around the world to increase the number of transferable bovine embryos and to improve the viability of these embryos after ET into recipients. Treatment of donor cows with bovine somatotropin (bST) has given satisfactory results that have increased the hopes for an improved commercial production of embryos (Moreira et al. 2002 Theriogenology 57, 1371–1387). In order to demonstrate the effect of bST on the production of transferable embryos, degenerated embryos and non-fertilized oocites, 16 high-value genetic Brahman cows were synchronized and later subjected to a superovulatory (SO) treatment. The SO treatment consisted of decreasing doses of FSH (Folltropin-V®, Vetrepharm Inc, Ontario, Canada) every 12h for 4 days (2.5, 2.0, 1.5, 0.5×2/day). Forty-eight hours after the beginning of the SO treatment, a dose of PGF2α (Lutalyse®, Upjohn, Kalamazoo, MI, USA.) was given to all donors (i.m.). Estrus was detected by careful observation. At the time of first AI (6 hours after estrus detection) all cows were given (i.m. a GnRH (Buserelin, Conceptal®, Intervet, Boxmeer, The Netherlands) analogue and randomly assigned to one of the following groups: bST (n=8), which were treated with 500mg (s.c.) of bST (Boostin-S®, Schering-Plough Caracas, Venezuela) or control group (n=8) not treated. The second AI was done 10 hours after the first AI. Immediately after the uterine recovery, oocytes and embryos were evaluated and classified, according to their morphologic characteristics, into transferable embryos (TE) of good (TE1) or medium (TE2) quality, degenerated embryos (DE) and non-fertilized oocytes (NF). CL were evaluated by rectal palpation to estimate the ovulatory response. The statistical comparison between experimental groups (percentage of each category) was carried out using a chi-square test of SAS. Results are presented in Table 1. Results show that bST donor treatment increases the number and quality of transferable embryos. Key words: bST, ET, donor, embryo, superovulation. Table 1 Number and percentages of transferrable and degenerated embryos and non-fertilized oocytes in superovulated Brahman cows after treatment with bST