Development Of Assisted Reproductive Technologies Research Articles

Investigating media that support red wolf (Canis rufus) sperm viability and capacitation in vitro.

SummaryThe red wolf is a critically endangered canid, with ~250 and ~20 individuals in the ex situ and reintroduced wild populations, respectively. Assisted reproductive technologies such as sperm cryopreservation and in vitro fertilization therefore represent critically-needed tools to manage these populations. However, the motility of post-thaw red wolf sperm rapidly declines during in vitro incubation, hindering the ability to develop these technologies. In this study, we evaluated the influence of several culture media (a modified canine capacitation medium (mCCM), a modified North Carolina State University-23 medium (mNCSU-23), a synthetic oviductal fluid (SOF), a fertilization Tyrode’s medium base or Fert-TALP (FERT), and a TRIS-based buffer (TRIS)) on the survival and capacitation of red wolf sperm during extended (18 h) incubation at 38.5°C and 5% CO2. Red wolf sperm motility averaged (±s.e.m.) 73.8 ± 7.1% at the time of collection, and was better maintained over 4 h incubation in mCCM (55.0 ± 9.8%) and mNCSU-23 (54.7 ± 10.4), compared to mSOF (43.8 ± 8.3%), FERT (30 ± 10.5), and TRIS (16.4 ± 4.1%) solutions. Patterns of tyrosine phosphorylation signal, as assessed via immunocytochemistry, indicated induction of capacitation between 2 and 4 h in vitro culture. Tyrosine phosphorylation signal was particularly robust in mCCM and mNCSU-23 incubated sperm, although significant acrosome exocytosis was not observed in response to progesterone supplementation after 3 h incubation in any of the media. In sum, results indicate mCCM and mNCSU-23 are promising base media for the in vitro incubation and capacitation of red wolf sperm, for assisted reproduction applications.Lay summaryDevelopment of assisted reproductive technologies such as in vitro fertilization and artificial insemination is of high importance to the genetic management of critically endangered species such as the red wolf (Canis rufus). However, these technologies require the ability to maintain sperm viability and function during extended incubation, which has not been successful for the red wolf thus far. In this study, various culture media developed for sperm/egg/embryo culture in large mammalian species were evaluated for their ability to maintain red wolf sperm motility under physiological incubation conditions. Media and conditions previously utilized for domestic dog sperm were found to best support sperm incubation and capacitation (process of becoming competent to fertilize an egg) in the red wolf, representing a key step for future development of assisted reproductive technologies for the species.

Kliniğimizde doğum yapan ikiz gebeliklerin değerlendirilmesi: Üç yıllık deneyim

Objective: Twin pregnancies constitute 1–2% of all pregnancies. With the recent developments in assisted reproductive technologies, the incidence of multiple pregnancy has increased. Preterm labor is held responsible for the poor neonatal outcomes primarily. In our study, we aimed to assess fetal-maternal outcomes of twin pregnancies seen in our clinic. Methods: One hundred and thirty twin pregnancy cases out of 4241 pregnant women who delivered in our clinic between 01.01.2017 and 01.01.2020 were included in the study. The labor records of the patients were reviewed retrospectively and their data for age, week of gestation, delivery type, birth weight, fetal sex, chorionicity characteristics and laboratory parameters were recorded. Definitive statistics and SPSS 21.0 for statistical analyses were used to evaluate the data obtained from the study. The data were presented as mean ± SD (standard deviation). Results: The incidence of twin pregnancy was found 3%. In the ultrasonographic imaging evaluated during diagnosis, 27.7% of the cases were monochorionic and 72% of them were dichorionic. Of the pregnant women, 12.3% were at term and 87.7% were at preterm period. When the preterm fetuses were evaluated, 37.7% of 114 preterm fetuses were delivered at late preterm period, 29.2% of them at mid-preterm period and 20.8% at premature preterm period. The rates of treatment pregnancy (pregnancy provided by ovulation induction and assisted reproductive technologies) and spontaneous twin pregnancy were 20% and 80%, respectively. Mean maternal age was found 31.3. When evaluated in terms of maternal morbidity, preterm labor and premature rupture of membrane were the most common problems. Mean newborn weight was 1832g and 59.3% of the newborns had low birth weight and 21.8% of them had very low birth weight. While one fetus had transverse presentation in 12.5% of the patients, at least one fetus had breech presentation in 53.1% of the cases and 34.4% of the cases had head-head presentation. Conclusion: Twin pregnancies are characterized by the increased feto-maternal risks. Therefore, both antenatal and intrapartum management should be maintained diligently.

Assisted reproductive technologies: psychoneurological, moral-ethical, and socio-cultural aspects

The paper provides a definition of sexual and reproductive health and infertility and also reflects modern ideas about ways to overcome infertility using assisted reproductive technologies, such as in vitro fertilization (IVF) and surrogacy. It shows the specificity of the impact of an IVF procedure on the mental health of a potential mother. The features of the neonatal health status, as well as neuropsychiatric disorders in babies born using the IVF procedure are described. The authors present two types of surrogacy (traditional and gestational ones) and the features of their use in different countries according to governmental legislative regulation, socioeconomic and religious factors, and cultural traditions in society. They unveil the features of a psychological relationship between the mother (surrogate and presumed one) and the fetus. The consequences of surrogacy for a surrogate mother, genetic parents, and a child himself/herself are noted to be little studied. It is shown that the development of assisted reproductive technologies (IVF and surrogacy), on the one hand, helps fight infertility and, on the other hand, entails a number of problems (moral and ethical, legal, cultural and religious, socioeconomic, and neuropsychiatric ones) that need to be solved in order to prevent psychological, neurological, and mental abnormalities in all the participants (a surrogate mother, an unborn child, and potential parents) in the assisted reproductive process:

Development of assisted reproductive technologies for Mus spretus†.

The genus Mus consists of many species with high genetic diversity. However, only one species, Mus musculus (the laboratory mouse), is common in biomedical research. The unavailability of assisted reproductive technologies (ARTs) for other Mus species might be a major reason for their limited use in laboratories. Here, we devised ARTs for Mus spretus (the Algerian mouse), a commonly used wild-derived Mus species. We found that in vitro production of M. spretus embryos was difficult because of low efficacies of superovulation with equine chorionic gonadotropin or anti-inhibin serum (AIS) (5-8 oocytes per female) and a low fertilization rate following in vitro fertilization (IVF; 15.2%). The primary cause of this was the hardening of the zona pellucida but not the sperm's fertilizing ability, as revealed by reciprocal IVF with laboratory mice. The largest number of embryos (16 per female) were obtained when females were injected with AIS followed by human chorionic gonadotropin and estradiol injections 24h later, and then by natural mating. These in vivo-derived 2-cell embryos could be vitrified/warmed with a high survival rate (94%) using an ethylene glycol-based solution. Importantly, more than 60% of such embryos developed into healthy offspring following interspecific embryo transfer into (C57BL/6×C3H) F1 female mice. Thus, we have devised practical ARTs for Mus spretus mice, enabling efficient production of embryos and animals, with safe laboratory preservation of their strains. In addition, we have demonstrated that interspecific embryo transfer is possible in murine rodents.

Profiling the DNA methylation patterns of imprinted genes in abnormal semen samples by next-generation bisulfite sequencing.

Changes in DNA methylation modifications have been associated with male infertility. With the development of assisted reproductive technologies (ARTs), abnormal DNA methylation in sperm, especially in imprinted genes, may impact the health of offspring and requires an in-depth study. In this study, we collected abnormal human semen samples, including asthenospermic, oligospermic, oligoasthenospermic and deformed sperm, and investigated the methylation of imprinted genes by reduced representation bisulfite sequencing (RRBS) and bisulfite amplicon sequencing on the Illumina platform. The differentially methylated regions (DMRs) of imprinted genes, including H19, GNAS, MEG8 and SNRPN, were different in the abnormal semen groups. MEG8 DMR methylation in the asthenospermic group was significantly increased. Furthermore, higher methylation levels of MEG8, GNAS and SNRPN DMR in the oligospermic and oligoasthenospermic groups and a decrease in the H19 DMR methylation level in the oligospermic group were observed. However, the methylation levels of these regions varied greatly among the different semen samples and among individual sperm within the same semen sample. The SNP rs2525883 genotype in the H19 DMR affected DNA methylation. Moreover, DNA methylation levels differed in the abnormal semen groups in the non-imprinted genomic regions, including repetitive sequence DNA transposons and long/short interspersed nuclear elements (LINEs and SINEs). Our study established that imprinted gene DMRs, such as H19, GNAS, SNRPN and MEG8, were differentially methylated in the abnormal semen groups with obvious inter- and intra-sample heterogeneities. These results suggest that special attention needs to be paid to possible epigenetic risks during reproduction.

The Porcine Experimental Model for Oocyte Cryopreservation by Vitrification

Pigs have anatomical-physiological similarities with the human in several aspects, mainly in the reproduction function, which is of great interest in this review. However, in oocytes, there are differences between both species, impacting the vitrification success. For example, porcine oocytes have higher intracellular lipid content than women oocytes, affecting cryoprotectant permeability and increasing ice crystal formation. Despite these differences, the porcine model has been important for the development of assisted reproduction technologies in humans, as well as in the genetic improvement of livestock.

Male Infertility is a Women's Health Issue-Research and Clinical Evaluation of Male Infertility Is Needed.

Infertility is a devastating experience for both partners as they try to conceive. Historically, when a couple could not conceive, the woman has carried the stigma of infertility; however, men and women are just as likely to contribute to the couple’s infertility. With the development of assisted reproductive technology (ART), the treatment burden for male and unexplained infertility has fallen mainly on women. Equalizing this burden requires reviving research on male infertility to both improve treatment options and enable natural conception. Despite many scientific efforts, infertility in men due to sperm dysfunction is mainly diagnosed by a semen analysis. The semen analysis is limited as it only examines general sperm properties such as concentration, motility, and morphology. A diagnosis of male infertility rarely includes an assessment of internal sperm components such as DNA, which is well documented to have an impact on infertility, or other components such as RNA and centrioles, which are beginning to be adopted. Assessment of these components is not typically included in current diagnostic testing because available treatments are limited. Recent research has expanded our understanding of sperm biology and suggests that these components may also contribute to the failure to achieve pregnancy. Understanding the sperm’s internal components, and how they contribute to male infertility, would provide avenues for new therapies that are based on treating men directly for male infertility, which may enable less invasive treatments and even natural conception.

Fertility preservation options for transgender individuals.

Gender affirming medical and surgical treatments affect the reproductive potential of transgender individuals. Prior to the development of assisted reproductive technologies (ART), genital gender-affirming surgery frequently eliminated a patient’s reproductive potential. Today, all patients should be counseled on their fertility preservation (FP) options before medical and surgical transition, yet this appears to seldom occur in practice. The following review is the result of a systematic literature search of PubMed, Medline and Google Scholar to identify current and future FP options, barriers to treatment patients face, practice patterns of transgender health care providers, and if there were any standardized counseling protocols. Options for transwomen at any point in their transition range from simply providing a semen sample to be used with assistive reproductive techniques to experimental techniques involving testicular cryopreservation followed by in vitro initiation of spermatogenesis. Transmen before and after starting hormone therapy can pursue any assistive reproductive techniques available for ciswomen. Future options currently under investigation include ovarian tissue cryopreservation (OTC) with in vitro oocyte maturation. In addition to counseling about their FP options, patients should be advised prospectively about the requirements, process details, the total costs associated with achieving pregnancy, and the inherent risks associated with using preserved genetic material including risk of failure, and maternal and fetal health risks. Transgender patients report using assistive reproductive services difficult, due to a lack of dialogue about fertility and the lack of information offered to them- presumably because their circumstances do not fit into a traditional narrative familiar to providers. Physicians and health care providers would benefit from better educational tools to help transgender patients make informed decisions and better training about transgender patients in general, and FP options available to them.

Implications of Assisted Reproductive Technologies for Pregnancy Outcomes in Mammals.

Development of assisted reproductive technologies has been driven by the goals of reducing the incidence of infertility, increasing the number of offspring from genetically elite animals, facilitating genetic manipulation, aiding preservation and long-distance movement of germplasm, and generating research material. Superovulation is associated with reduced fertilization rate and alterations in endometrial function. In vitro production of embryos can have a variety of consequences. Most embryos produced in vitro are capable of establishing pregnancy and developing into healthy neonatal animals. However, in vitro production is associated with reduced ability to develop to the blastocyst stage, increased incidence of failure to establish pregnancy, placental dysfunction, and altered fetal development. Changes in the developmental program mean that some consequences of being produced in vitro can extend into adult life. Reduced competence of the embryo produced in vitro to develop to the blastocyst stage is caused largely by disruption of events during oocyte maturation and fertilization. Conditions during embryo culture can affect embryo freezability and competence to establish pregnancy after transfer. Culture conditions, including actions of embryokines, can also affect the postnatal phenotype of the resultant progeny.

Development of assisted reproductive technologies in small animal species for their efficient preservation and production.

Assisted reproductive technologies (ARTs) are widely used in the animal industry, human clinics, and for basic research. In small laboratory animal species such as mice, ARTs are essential for the production of animals for experiments, the preservation of genetic resources, and for the generation of new strains of genetically modified animals. The RIKEN BioResource Research Center (BRC) is one of the largest repositories of such animal bioresources, and maintains approximately 9,500 strains of mice with a variety of genetic backgrounds. We have sought to devise ARTs specific to the reproductive and physiological characteristics of each strain. Such ARTs include superovulation, in vitro fertilization (IVF), the cryopreservation of embryos and spermatozoa, transportation of cryopreserved materials and embryo transfer (ET). Of these, superovulation likely has the most influence on animal production because it determines the quantity of starting material for other ARTs. Superovulation using anti-inhibin serum combined with estrous synchronization has resulted in approximately a three-fold increase in production efficiency with IVF–ET in the C57BL/6J strain. Wild-derived strains are important as genetically diverse resources for murine rodents (Genus Mus), and many are unique to the BRC. We have also successfully developed ARTs for more than 50 wild-derived strains, which have been cryopreserved for future use. Our work to improve and develop ARTs for mice and other small laboratory species will contribute to the cost-effectiveness of routine operations at repository centers, and to the provision of high quality animals for research use.

Kwestia prokreacji w prawosławiu

W Kościele prawosławnym prokreacja jest postrzegana nie tylko jako aktywność ludzka mająca na celu przedłużenie gatunku, ale również jako dar Boga, Który jest absolutnym i jedynym Władcą życia człowieka. Zrozumienie prokreacji, z chrześcijańskiego punktu widzenia, wymaga odniesienia do sakramentu małżeństwa. W ramach bowiem małżeństwa sakramentalnego, które jest monogamiczne, heteroseksualne i pobłogosławione w Kościele, popęd seksualny podlega transformacji. Poczęcie i zrodzenie potomstwa jest owocem miłości obojga rodziców. Początek istnienia duszy i ciała każdej osoby ludzkiej dokonuje się równocześnie. Liczne świadectwa pochodzące z tradycji kanonicznej, liturgicznej i patrystycznej chrześcijaństwa wschodniego dowodzą, iż od pierwszych wieków aborcja była traktowana jako zabójstwo i grzech ciężki, wymagający długotrwałej pokuty. Współcześnie Kościół prawosławny podejmuje wyzwania związane z rozwojem technologii wspomagania rozrodu. W oficjalnych dokumentach prawosławnych podkreśla się, że podejmowanie decyzji skorzystania ze wspomaganej prokreacji powinno uwzględniać zarówno nauczanie doktrynalne, jak i sytuację egzystencjalną danej pary małżeńskiej. Szczegółowe zalecenia dotyczą pozaustrojowego zapłodnienia in vitro. Prawosławni bioetycy wypowiadają się przeciwko stosowaniu technik klonowania oraz eksperymentom z komórkami ludzkimi z grupy zarodkowych.

Comprehensive analysis of whole genome methylation in mouse blastocysts cultured with four different constituents following in vitro fertilization

BackgroundWith the development of assisted reproductive technology (ART), diseases believed to be caused by ART have begun to be identified as imprinted disease. However, no conclusion has been reached. So we sought to determine whether ART procedures disturb gene methylation and whether imprinted genes alone are selectively disturbed. To examine whether the constituents of the culture medium contribute to the changes in methylation, we used a mouse model to conduct IVF and comprehensively analyzed 5′–C–phosphate–G–3′ (CpG) by reduced representation bisulfite sequencing (RRBS) using a second-generation sequencer to determine changes in methylation using four types of culture media with different amino acid constituents.ResultsWe cultured ova to the blastocyst stage in a mouse model in culture media with four different amino acid constituents. Each culture medium included (1) KSOM culture medium (NoAA), (2) KSOM media + essential amino acids (EAAs), (3) KSOM medium + non-essential amino acids (NEAAs), or (4) KSOM medium + EAAs + NEAAs (AllAA) analyzed by reduced representation bisulfite sequencing. The results showed that (1) there were many regions that maintained hypermethylation with NEAAs, (2) there was little effect of demethylation on reprogramming in the 5′UTR and promoter regions, and (3) specific changes were observed in imprinted genes such as Nnat and Nespas.ConclusionsCompared with EAAs, NEAAs could protect genes from demethylation caused by reprogramming. On the imprinted genes, methylation of the promoter region of H19 was decreased by NEAAs, suggesting that specific genes were prone to changes in methylation. It was suggested that these changes could provide similar results in humans. Further studies are needed to understand how changes in methylation may affect gene expression profiles.

Tauroursodeoxycholic acid (TUDCA) enhanced intracytoplasmic sperm injection (ICSI) embryo developmental competence by ameliorating endoplasmic reticulum (ER) stress and inhibiting apoptosis.

The objective of this study was to examine the effect of tauroursodeoxycholic acid (TUDCA) on intracytoplasmic sperm injection (ICSI) embryos by evaluating endoplasmic reticulum (ER) stress, apoptosis, and embryo developmental competence in vitro and in vivo. ER stress-associated genes and apoptosis-associated genes were measured and apoptosis index was analyzed. Embryo developmental competence was assessed in vitro and in vivo via the inner cell mass (ICM)/trophectoderm (TE) index, pregnancy and implantation rates, and birth rate. The relative mRNA and protein expression of binding immunoglobulin protein (BIP) was significantly higher in the ICSI embryo group without TUDCA treatment (ICSI-C) than in the in vitro fertilization (IVF) group and in the ICSI embryo group with TUDCA treatment (200 μM) (ICSI-T), while TUDCA ameliorated ER stress in ICSI embryos. Embryos in the ICSI-C group showed a higher apoptosis index than those in the IVF group and ICSI-T group, and there was no significant difference between the IVF group and ICSI-T group. TUDCA can significantly improve ICSI embryo developmental competence in vitro and in vivo based on the ICM/TE index, pregnancy and implantation rates, and birth rate. ICSI embryos manifested high ER stress and high apoptosis, while TUDCA ameliorated ER stress and reduced apoptosis in ICSI embryos. TUDCA can significantly improve the developmental competence of ICSI embryos in vitro and in vivo. This study provides a new idea for improving the efficiency of ICSI, and it will also have a positive effect on the development of assisted reproduction technologies for humans and other animals.

229 From head to tail: A red wolf sperm project

Development of assisted reproductive technologies for the critically endangered red wolf (Canis rufus) is crucial to the maintenance of genetic diversity to support species recovery. Towards this goal, a cryopreservation protocol has previously been developed for red wolf sperm; however, the ability of the gametes to undergo capacitation has not been assessed in this species. Previously, we have shown that oviductal extracellular vesicles (oEVs) improve cat sperm motility and fertilizing ability. The objectives were to (1) compare the effects of culture media on motility and acrosomal integrity of fresh sperm, and select the best medium that can be used in a capacitation protocol; (2) identify potential biomarkers for sperm cryo-tolerance; and (3) determine the influence of canine oEVs on sperm survival and motility post-thaw. In Study 1, sperm were collected by electro-ejaculation from adult red wolves (n=8) and immediately cryopreserved in TRIS-egg yolk buffer with 8% glycerol or incubated for 18h in 5% CO2 and 38.5°C in one of the following media: canine capacitation medium (CCM), FERT-TALP (FERT), NCSU, synthetic oviductal fluid (SOF) and TRIS. At 0, 1, 2, 3, 4, and 18h, sperm were evaluated for total motility and acrosome integrity (FITC-PNA). In Study 2, sperm with high (>80%, HM, n=2 wolves) and low (<15%, LM, n=2 wolves) motility post-incubation at 4°C in the cryopreservation medium for 18h were subjected to proteomic analysis. In Study 3, oviducts were collected from domestic dogs (1-9 years, n=12) after elective spaying, and oEVs from various stages of the oestrous cycle [early follicular (EF), late follicular (LF), early luteal (EL), and late luteal (LL)] were isolated using the Total Exosome Isolation kit (Invitrogen). Frozen-thawed red wolf sperm (n=4 males) were incubated with 30×106 oEVs in non-capacitating CCM, and assessed as in study 1 at 0, 0.5, 1, 2, 3, 4, 6, 8, and 10h. Data were analysed using a paired samples t-test with 95% CI (Prism8, GraphPad Inc.). Sperm incubated in CCM and NCSU had higher motility than those in FERT, SOF, and TRIS after 2h of incubation and onward (2 h: 65±6, 68±6, 42±10, 57±8, and 43±5; 3 h: 60±9, 63±8, 36±11, 46±9, and 34±6; 4 h: 60±9, 60±10, 30±10, 43±8, and 20±5; 18 h: 12±7, 15±7, 9±5, 3±2, and 0, respectively; P<0.05). After 1h of incubation, samples incubated in CCM, NCSU, and SOF had a higher number of sperm with intact acrosomal membranes (P>0.05) than other treatments. A total of 179 proteins were identified, of which 129, including those regulating energy metabolism and mitochondrial mediated apoptosis, were differentially expressed between HM and LM. Preliminary data from Study 3 suggested that thawing and incubating sperm in the presence of LF, EL, and LL oEVs improved sperm motility. In conclusion, CCM and NCSU sustained sperm survival after invitro incubation and could be candidates for invitro fertilization studies in the red wolf. Data generated from sperm proteomic analysis provided insights into cellular pathways regulating sperm cryo-sensitivity. Finally, we demonstrated the potential of oEVs in improving wolf sperm survival post-thawing.

Hormonal induction of spermiation in a Eurasian bufonid (Epidalea calamita)

BackgroundAmphibian diversity is declining at an alarming rate due to habitat loss, invasive species, climate change and diseases. Captive assurance colonies have been established for some species at risk; however, many species do not breed well in captivity and the development of assisted reproductive technologies (ART) is critical to help sustain genetic diversity. To date, the majority of the work has been accomplished in species from the American continent and Australia, and there is a need to address similar breeding challenges in Eurasian and African species of amphibians.MethodsThe aim of this study was to develop a hormone protocol for stimulation of spermiation in Epidalea calamita as a model for Eurasian bufonids. Hence, the effect on sperm production and quality of three doses of chorionic gonadotropin hormone (5, 10 and 15 IU hCG/g) over time (1 to 24 h) was evaluated. In addition, cold storage (at 5 °C) of sperm for 24 and 48 h and three frequencies for hormonal treatment (weekly, biweekly and monthly) were examined.ResultsHormone concentrations of 10 or 15 IU of hCG induced spermiation in 100% of males and produced sperm of comparable quality, while 5 IU hCG stimulated spermiation in only 40% of males. Total motility peaked between 1 to 4 h post-treatment with 10 IU hCG, whereas treatment with 15 IU hCG peaked between 2 to 6 h. After 24 h of cold storage total motility dropped by 20% and forward motility dropped by 10% for both the 10 and 15 IU treatments. Weekly hormone administration resulted in higher variation between trials in all motility parameters and a lower overall Total Motility and Forward Movement. Furthermore, the effect of exogenous hormone treatment overlapped between the last two trials in the weekly frequency. Sperm concentration was higher in the first trial for all frequencies but showed no differences among other trials.ConclusionsOverall, these results show that hormone concentration, time after treatment, frequency of hormone treatment and cold storage should be borne in mind when developing a hormone stimulation protocol for Eurasian amphibian species.

Perinatal complications and live-birth outcomes following assisted reproductive technology: a retrospective cohort study.

BackgroundWith the development of assisted reproductive technology (ART) and its increasing success rate in the mainland of China, more attention has been paid to the safety of ART. In this study, we explored the associations between conception by ART and pregnancy/perinatal complications, and neonatal outcomes compared with similar outcomes following spontaneous conception.MethodsThis retrospective cohort study of pregnancies over a 3-year period (2013–2015) was performed at Beijing Obstetrics and Gynecology Hospital, Beijing, China. Subjects were divided into two groups: conception by ART (n = 2256) or spontaneous conception (n = 6768). According to different fertilization modes, the ART group was divided into in vitro fertilization (IVF, n = 1873) and intracytoplasmic sperm injection (ICSI, n = 383) subgroups. The ART group was also divided into two different embryo transfer methods; fresh embryo transfer (ET, n = 1583) and frozen embryo transfer (FET, n = 673) subgroups. Pregnancy complications, perinatal complications, and neonatal outcomes of the enrolled subjects were investigated and analyzed by univariate analysis and multivariate logistic regression.ResultsAfter adjustment for maternal age, gravidity, parity, maternal education, smoking, alcohol consumption, and body mass index (BMI), pregnancies conceived by ART were associated with a significantly increased incidence of gestational diabetes mellitus (GDM; OR 1.88, 95% CI 1.56–2.27), gestational hypertension (OR 2.18, 95% CI 1.83–2.60), and intrahepatic cholestasis of pregnancy (ICP) (OR 2.79, 95% CI 2.15–3.64), compared with spontaneous conception. These associations were similar for the singleton group. In the twin group, only the incidence of ICP was significantly higher than in controls. We found that pregnancies conceived by ART were associated with perinatal complications, including placental abruption (OR 2.14, 95% CI 1.33–3.45), premature rupture of membranes (PROM; OR 1.24, 95% CI 1.06–1.45), postpartum hemorrhage (OR 2.89, 95% CI 2.33–3.59) and polyhydramnios (OR 2.01, 95% CI 1.29–3.16). The singleton group had a similar result with placental abruption, but not with fetal membranes ruptures before labor and polyhydramnios. There were no significant differences in the incidence of these perinatal complications in the twin group. Some neonatal outcomes, including preterm labor (OR 4.29, 95% CI 3.84–4.80) and low birth weight (OR 1.72, 95% CI 1.42–2.08), were more likely to occur with singleton births after ART. However, there were no significant differences for these outcomes from twin pregnancies. Perinatal complications and neonatal outcomes were consistent between the IVF and ICSI subgroups. The FET and ET subgroups showed a similar increase in complications, except for the incidence of placental abruption. After taking into account the effects of parity, birth plurality and maternal age, the ART group still exhibited increased maternal and neonatal complications, although some differences narrowed or disappeared.ConclusionsThis retrospective cohort study demonstrated that patients who underwent ART were at increased risk of several adverse pregnancy outcomes compared with women who conceived spontaneously. These complications may be attributed in part to the relatively high multiple pregnancy rate after ART. Elective single embryo transfer should be promoted in China to reduce the obstetrical risks of ART pregnancy. Singletons of ART pregnancy exhibited increased maternal and neonatal complications as well, suggesting that underlying infertility or other maternal or parental factors may contribute to the adverse outcomes.

Assisted reproductive technology strategies in uterus transplantation

The development of assisted reproductive technology (ART) through four decades has led to offer the ultimate treatment for nearly all forms of infertility. The only remaining factor of childlessness however that still eludes ART and its routine variants are the absolute uterine infertility factors, for which the only option is an experimental approach, uterus transplantation. Progresses has been made over the past few years, and more are underway for simplifying the process notably for simplifying the uterus extraction step performed in the uterus donor. Furthermore, as the technique is being better mastered, the original indications for uterus transplantation, the congenital or acquired absence of the uterus, are now widened to also include incurable uterine fibrosis, or Asherman's syndrome. The ART-related practicalities of uterus transplantation, ovarian stimulation and uterine priming are being discussed in the present review.

Comparative reproductive function in cervids: implications for management of farm and zoo populations

The cervids represent a complex assemblage of taxa characterized by extreme diversity in morphology, physiology, ecology and geographical distribution. Farmed species (for example red deer and fallow deer) are usually the common larger-bodied, gregarious and monotocous species that express marked reproductive seasonality in their temperate environment. Their commercial importance has facilitated considerable research into reproductive physiology and the development of assisted reproductive technologies (ART). In contrast, the remaining species, including many of tropical origin, show wide diversity in reproductive patterns, have generally received little scientific scrutiny, and include a number of endangered taxa that are reliant on ex situ conservation efforts (such as captive breeding) to ensure their survival. Domestication and ex situ management programmes have been associated with widespread translocation of various cervid species around the world, often placing the animals in environments that are not compatible with their evolved reproductive patterns. For example, the summer calving/lactation pattern of red deer, attuned to northern continental climatic patterns, is frequently misaligned with seasonal changes in feed availability in the Australasian pastoral environment. Similarly, seasonal or aseasonal calving patterns of tropical species translocated to temperate regions are usually associated with increased perinatal mortality of calves born in cool seasons. Conversely, temperate species in tropical zones may exhibit aberrant reproductive patterns in the absence of biologically significant photoperiod fluctuations. ARTs, which presently include artificial insemination, embryo transfer and in vitro embryo production, have potential application to the genetic management and population growth of various cervid species. Although application to some farmed cervid species is widespread, these technologies are rarely directly transferable from farmed to endangered species. Even within species, ART protocols developed successfully for one genotype (i.e. subspecies) may be ineffective in another (for example superovulation of red deer and wapiti). Therefore, application to genetic management of endangered species necessitates prior research into their reproductive patterns. This is often difficult because of the rarity of the animals, a lack of suitable handling facilities for the particular species, and the timid nature of the deer. More recently, however, non-invasive reproductive profiling, based on remote collection and monitoring of excreted steroid metabolites, has facilitated such research.

First cryopreservation of phyllostomid bat sperm

Bats, the second-largest mammalian order, are widely distributed and provide crucial ecosystem services. Their reproductive biology comprises noteworthy characteristics, such as long-term sperm storage, delayed implantation or even delayed fetal development. The understanding of these mechanisms remains limited. Research in reproductive biology may become crucial for the conservation of endangered bat species. Indeed, the IUCN Red List of Threatened Species currently lists 76 bat species as endangered or critically endangered, for which conservation measures should be taken. The development of assisted reproductive technologies (ART) in bats would thus open new perspectives.In this context we here describe the first successful cryopreservation of bat sperm in the Seba’s short-tailed bat (Carollia perspicillata). Epididymal sperm of 30 males was pooled in 6 sample sets and diluted using four different cryo-diluents: Canifreeze, modified Canifreeze, BotuCrio® and Test egg yolk extender. Glycerol and a mixture of glycerol and methylformamide were used as cryoprotectants. Best post-thaw results were achieved when sperm was diluted in Test egg yolk extender containing a mixture of glycerol and methylformamide as cryoprotectant. Post-thaw sperm motility, progressive motility and acrosome integrity of 42.0 ± 3.4%, 28.3 ± 6.3% and 87.7 ± 1.7%, respectively were superior to all other variants tested. It remains to be determined if the post-thaw quality of cryopreserved bat spermatozoa achieved in this study is sufficient for the use in artificial inseminations and produces acceptable pregnancy rates. Successful cryopreservation of spermatozoa in C. perspicillata may serve as the first model to establish gamete rescue programs and develop ART in other, critically endangered bat species.

98 Efficacy of commercial equine semen freezing extenders for cryopreservation of southern white rhinoceros (Ceratotherium simum simum) sperm

Once nearly extinct in the wild, the southern white rhinoceros is currently listed as near threatened by IUCN. This status is likely to change as poaching continues to escalate. To preserve the species’ current genetic diversity, cryopreserving and biobanking white rhinoceros sperm is imperative. The horse is the closest domestic relative of the rhinoceros and a useful model for the development of assisted reproductive technologies, including semen cryopreservation. Two equine semen cryopreservation protocols were compared to a common rhinoceros freezing method. Semen was collected from a single male on 3 occasions by electroejaculation. Initial semen parameters were 86% motility; speed 3.2 (scale 1-5); 89% plasma membrane integrity; and 95% intact acrosomes. Semen was extended 1:1 in INRA 96 (IMV Technologies, L’Aigle, France) before centrifugation at 400×g for 10min. Supernatant was removed and the sperm pellet was subjected to 1 of 2 treatments: resuspension in 500µL of either BotuCrio (Botupharma, Botucatu, Brazil) or Cryomax (ARS Inc., Chino, CA, USA), both containing a proprietary combination of glycerol and an amide as cryoprotectants. Following a 40-min cool at 4°C, extended semen was frozen in vials at a cooling rate of 30°C/min for 3min before LN submersion. Control semen was extended 1:1 in TEST-Y buffer without cryoprotectant and cooled for 2.5h before adding glycerol to a final concentration of 4%. Extended sperm (500µL) was frozen in vials at 12.5°C/min for 15min before LN submersion. Initial motility score (IMS;% motile×speed of progression2), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded after extension. All vials were thawed at 37°C for 60s and the cryoprotectant was removed by centrifugation. Sperm pellets were resupended in M199+HEPES and sperm was evaluated for the characteristics described above at 37°C at 0, 30, and 60min (T0, T30, T60) post-thaw. All data are expressed as a percentage of initial (%IMS,%IPL, and%IAC) to account for the differences in sperm parameters between ejaculates. Cryopreservation protocol significantly affected%IMS at T0 (P=0.0131, Table 1). Although the differences were significant only at T0, sperm frozen in Botucrio or Cryomax tended to maintain a higher%IMS than the control freeze at all time points. However, sperm frozen in Cryomax lost a greater percentage of%IMS over time (67% from T0 to T60v. 44 and 46% for Botucrio and TEST-Y, respectively). Cryopreservation protocol did not affect%IAC or%IPL at any time point, but again Cryomax and Botucrio tended to be higher than TEST-Y. This study indicates that rhinoceros sperm may suffer less cryodamage in Botucrio or Cryomax frozen at 30°C/min than in the conventional TEST-Y frozen at 12.5°C/min. Table 1.Percent of initial motility score (IMS), plasma membrane integrity (IPL), and acrosome integrity (IAC) at 0, 30, and 60min post-thaw (T0, T30, and T60, respectively)