Homage to Professor Charles Thibault. Synthesis of the symposium and conclusions

"Reflexive biology", using the ambiguities of the term, is altogether a biology that plays with the mirrors of the different levels of organisation, which, endlessly, go from one place to another, organisms in their real environment to the genome via cells in vitro and others, but which is also reflexive because at any moment biology reflects the finalities and the worry of what it should serve.     

Transport of equine ovaries for assisted reproduction

Use of assisted reproduction to obtain foals from valuable mares post-mortem typically necessitates holding of ovaries during shipment to a laboratory. The present study evaluated whether holding ovaries briefly at a warm ( approximately 30 degrees C) temperature improves meiotic and developmental competence of oocytes, as determined after maturation in vitro and intracytoplasmic sperm injection. Ovaries were packaged in pairs in insulated containers, and held either at 24 or 25-35 degrees C for 4h, followed by cooling. Ovaries in both treatments were held for either a short (mean, 7-7.4h) or long (mean, 20.6-20.7h) duration before oocyte recovery. Control ovaries were collected en masse at the abattoir. The ovary temperature in this treatment slowly decreased to approximately 27 degrees C; oocyte recovery was performed after 3.5-7h total holding. There was no effect of temperature on oocyte meiotic or developmental competence within either treatment time period. Oocytes in the short duration holding group had similar meiotic competence to controls, but had a significantly decreased rate (P<0.05) of blastocyst development. Oocytes in the long duration holding group had decreased (P<0.05) meiotic competence and blastocyst development compared to controls. These findings indicate that storage of equine ovaries for only 7h may decrease blastocyst development, and that longer storage reduces both rate of oocyte maturation and blastocyst development. Further work is needed to determine if there is a critical time before 7h post-mortem by which equine oocytes should be recovered to maximize developmental competence.     

Production of embryos by assisted reproduction in the horse

In vitro embryo production is not yet successful in the horse, largely due to low rates of fertilization in vitro. However, methods to produce embryos from isolated oocytes have been developed. Oocytes may be recovered from living mares by aspiration of the dominant preovulatory follicle by trans-abdominal puncture, and from both preovulatory and immature follicles by trans-vaginal ultrasound-guided puncture. Transfer of in vivo-matured oocytes to the oviducts of bred recipient mares has resulted in good pregnancy rates (75-85%). Little work has been done on transfer of horse oocytes matured in vitro. Recovery rates of immature oocytes from mares in vivo are lower than those for cattle. In addition, work on oocytes recovered from horse ovaries post-mortem has shown that horse oocytes from smaller (< 20 mm diameter) viable follicles may not yet be meiotically competent. Methods for in vitro fertilization and for obtaining adequate numbers of competent immature oocytes from the mare must be developed before in vitro embryo production can become a useful clinical and research procedure in the horse.     

Mitochondrial DNA variations in ova and blastocyst: Implications in assisted reproduction

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission.     

Pharmacokinetics of the progesterone-containing vaginal tablet and its use in assisted reproduction

Natural progesterone, which is devoid of androgenic activity, is widely used in assisted reproduction for luteal and pregnancy support. The vaginal route has become the most established way to deliver natural progesterone because it is easily administered, avoids liver first-pass metabolism, and has no systemic side-effects. The vagina has a large potential for absorption, and through the 'uterine first-pass effect' vaginal administration results in higher uterine progesterone concentrations. We have investigated the pharmacokinetics of natural progesterone in the form of a vaginal tablet. A single dose of 100 mg resulted in a mean C(max) of 31.53 +/- 9.15 nmol/l with a T(max) of 6.92 +/- 3.12 h. The terminal half-life was 16.39 +/- 5.25 h. The pharmacokinetic data are discussed in relation to dose, age, and estrogen priming. Single-dose pharmacokinetics of 100 mg of progesterone vaginal tablets and gelatin capsules were evaluated over 24 h. Results indicated a similar mean T(max) of 6.92 +/- 3.12 and 6.23 +/- 6.57 h, respectively. However, a significantly higher C(max) was achieved by the vaginal tablet (31.95 +/- 9.15 and 23.85 +/- 9.57 nmol/l, respectively, P < 0.05). Continuous use of vaginal progesterone did not influence the hormonal, liver, or lipid profiles evaluated. There was no case of endometrial hyperplasia. The vaginal tablet was found to be well-tolerated, safe, and easily administered. In conclusion, progesterone-containing vaginal tablets have good pharmacokinetic properties and should be used for progesterone supplementation in IVF.     

Ovarian control for assisted reproduction in the domestic cat and wild felids

Of the 37 felid species, all but the domestic cat are classified as threatened with extinction in all or part of their native range. Additionally, the domestic cat is a valuable model for human biomedical research. Propagating some wild felids as well as domestic cat populations serving as human models is a major challenge primarily due to difficulties in transporting animals between facilities to ensure the pairing of genetically matched individuals, behavioral incompatibility between pairs and low fertility. Artificial insemination (AI) and in vitro fertilization/embryo transfer (IVF/ET) are powerful tools for helping manage rare populations. Developing successful assisted reproductive techniques requires knowledge of the female reproductive cycle and precise control of ovarian activity. Successful ovarian stimulation for AI and IVF/ET has been achieved in at least one-third of all cat species. However, sensitivity to a given gonadotropin treatment appears highly species-specific, and poor responses are common, particularly in felid species that exhibit spontaneous ovulations. Furthermore, current gonadotropin regimens have been demonstrated to perturb female reproductive function often leading to reduced fertility. Overall, ovarian response to exogenous hormonal stimulation has been highly variable, and pregnancy success after AI or IVF/ET remains low (<20%) in most species. Therefore, there is an immediate need to develop improved regimens that would allow more predictable ovarian responses in felids. We contend that recent research involving the use of progestins to control the ovary prior to gonadotropin stimulation shows promise for providing consistent ovarian stimulation in felids.     

Investigation of assisted fertilization and biology of reproduction by sperm microinjection

Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that under-lie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed.     

Conservation of wild animals by assisted reproduction and molecular marker technology

Wild animals are an integral component of the ecosystem. Their decimation due to abrupt natural calamities or due to gradual human intervention would be disastrous to the ecosystem and would alter the balance in nature between various biotic components. Such an imbalance could have an adverse effect on the ecosystem. Therefore, there is an urgent need to put an end to the ever increasing list of endangered species by undertaking both in situ and ex situ conservation using tools of modern biology, to ascertain the degree of genetic variation and reproductive competence in these animals. This review highlights the development and use of molecular markers such as microsatellites, minisatellites, mitochondrial control region, cytochrome b and MHC loci to assess the genetic variation in various Indian wild animals such as the lion, tiger, leopard and deer. The review also presents data on the semen profile of the big cats of India. Reproductive technologies such as cryopreservation of semen and artificial insemination in big cats are also highlighted.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

Seasonal variability in the fertilization rate of women undergoing assisted reproduction treatments

The objective of this study was to evaluate whether seasonality affects human-assisted reproduction treatment outcomes. For this, 1932 patients undergoing intracytoplasmic sperm injection (ICSI) were assigned to a season group according to the day of oocyte retrieval: winter (n = 435), spring (n = 444), summer (n = 469) or autumn (n = 584). Analysis of variance was used to compare the ICSI outcomes. The fertilization rate was increased during the spring (winter: 67.9%, spring: 73.5%, summer: 68.7% and autumn: 69.0%; p < 0.01). In fact, a nearly 50% increase in the fertilization rate during the spring was observed (odds ratio 1.45, confidence interval 1.20-1.75; p < 0.01). The oestradiol concentration per number of oocytes was significantly higher during the spring (winter: 235.8 pg/mL, spring: 282.1 pg/mL, summer: 226.1 pg/mL and autumn: 228.7 pg/mL; p = 0.030). This study demonstrates a seasonal variability in fertilization after ICSI, where fertilization is higher during the spring than at any other time.     

Magnetic activated cell sorting (MACS): utility in assisted reproduction

Assisted reproductive techniques (ART) have now been extensively incorporated in the management of infertile couples. But even after rapid methodological and technological advances the success rates of these procedures have been below expectations. This has led to development of many sperm preparation protocols to obtain an ideal semen sample for artificial reproduction. Sperm apoptosis has been heavily linked to failures in reproductive techniques. One of the earliest changes shown by apoptotic spermatozoa is externalization of phosphatidyl serine. Magnetic activated cell sorting (MACS) is a novel sperm preparation technique that separates apoptotic and non-apoptotic spermatozoa based on the expression of phosphatidylserine. This has led to the incorporation of MACS as a sperm preparation technique. The review highlights the principle and mechanism of this novel technique and enumerates its advantages as a sperm preparation technique. Its utility in ART as an efficient tool for sperm recovery and its application in cryopreservation of semen samples is also explained.     

The biology and methodology of assisted reproduction in deer mice (Peromyscus maniculatus)

Although laboratory-reared species of the genus Peromyscus—including deer mice—are used as model animals in a wide range of research, routine manipulation of Peromyscus embryogenesis and reproduction has been lagging. The objective of the present study was to optimize conditions for oocyte and/or embryo retrieval and for in vitro culturing. On average, 6.4 oocytes per mouse were recovered when two doses of 15 IU of pregnant mare serum gonadotropin (PMSG) were given 24 h apart, followed by 15 IU of hCG 48 h later. Following this hormone priming, females mated overnight with a fertile male yielded an average of 9.1 two-cell stage embryos. Although two-cell stage embryos developed to 8-cell stage in Potassium Simplex Optimized Medium (KSOM; Millipore-Chemicon, Billerica, MA, USA) in vitro, but not further, embryos recovered at the 8- to 16-cell stages developed into fully expanded blastocysts when cultured in M16 media in vitro. These blastocysts had full potential to develop into late stage fetuses and possibly into live pups. As a result of the present work, all stages of Peromyscus preimplantation development are now obtainable in numbers sufficient for molecular or other analyses. These advances provide the opportunity for routine studies involving embryo transfer (e.g., chimeras, transgenics), and preservation of genetic lines by cryopreservation.     

Endometrial claudin-4 and leukemia inhibitory factor are associated with assisted reproduction outcome

Background  

Claudin-4 (CLDN4) is one of several proteins that act as molecular mediators of embryo implantation. Recently, we examined immunolabeling of leukemia inhibitory factor (LIF) in the endometrial tissue of 52 IVF patients, and found that LIF staining intensity was strongly correlated with successful pregnancy initiation. In the same set of patients, we have now examined endometrial CLDN4 expression, to see how expression intensity may vary with LIF. We examined CLDN4 in the luteal phase of the menstrual cycle, immediately preceding IVF treatment. Our aim was to compare expression of LIF and CLDN4 in the luteal phase, and document these patterns as putative biomarkers for pregnancy.     

Leuprolide acetate reduces both in vivo and in vitro ovarian steroidogenesis in infertile women undergoing assisted reproduction

Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26-35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10(-12), 10(-9) and 10(-6)M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1=6479+/-3458; G2=3021+/-1119 ng/ml; p=0.04) and a lower testosterone peak in GC at 96h (G1=0.64+/-0.12 ng/ml; G2=0.50+/-0.19 ng/ml; P=0.02), but a higher fertilization rate (G1=67%; G2=83%; p=0.009). In vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10(-6)M; 606.0+/-114.3 ng/ml versus 1524.0+/-246.5 ng/ml; p=0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall.     

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations.     

Epigenetic heterogeneity of developmentally important genes in human sperm: Implications for assisted reproduction outcome

The molecular basis of male infertility is poorly understood, the majority of cases remaining unsolved. The association of aberrant sperm DNA methylation patterns and compromised semen parameters suggests that disturbances in male germline epigenetic reprogramming contribute to this problem. So far there are only few data on the epigenetic heterogeneity of sperm within a given sample and how to select the best sperm for successful infertility treatment. Limiting dilution bisulfite sequencing of small pools of sperm from fertile donors did not reveal significant differences in the occurrence of abnormal methylation imprints between sperm with and without morphological abnormalities. Intracytoplasmic morphologically selected sperm injection was not associated with an improved epigenetic quality, compared to standard intracytoplasmatic sperm injection. Deep bisulfite sequencing (DBS) of 2 imprinted and 2 pluripotency genes in sperm from men attending a fertility center showed that in both samples with normozoospermia and oligoasthenoteratozoospermia (OAT) the vast majority of sperm alleles was normally (de)methylated and the percentage of epimutations (allele methylation errors) was generally low (<1%). However, DBS allowed one to identify and quantify these rare epimutations with high accuracy. Sperm samples not leading to a pregnancy, in particular in the OAT group, had significantly more epimutations in the paternally methylated GTL2 gene than samples leading to a live birth. All 13 normozoospermic and 13 OAT samples leading to a child had <1% GTL2 epimutations, whereas one (7%) of 14 normozoospermic and 7 (50%) of 14 OAT samples without pregnancy displayed 1–14% GTL2 epimutations.