Fertilization and early embryology: Differential effect of epithelial cell-conditioned medium fractions on preimplantation mouse embryo development

Coculture studies using preimplantation embryos have led to a number of conflicting studies. In the human, ethical considerations have led to the preferential use of epithelial cell lines as distinct from human Fallopian tube cells. In an attempt to isolate factors influencing embryo development we have cultured 2-cell OF1 mouse embryos in media [Ménézo's B2 and Whittingham's T6 supplemented with vitamins and amino acids (T6VA)] conditioned on two types of kidney epithelial cells (MBDK and Vero). Different molecular weight fractions of conditioned medium were used to show the absence or presence of specific embryotrophic factors. With MDBK cells, B2 conditioned medium enhanced embryo development up to the blastocyst stage, while no blastocysts developed in B2 alone. When using T6VA medium, both the control and conditioned media showed a high percentage of blastocyst formation (57.0 and 54.0% respectively), while the different molecular weight fractions showed no added improvement. With Vero cells, B2 alone, B2 conditioned medium and fractions were all detrimental to embryo development. A high percentage of blastocyst formation (between 64.7 and 75.8%) was observed in T6VA alone, T6VA conditioned medium and fractions. Low blastocyst formation in a control medium can show strong positive results when medium is conditioned by cells. In contrast, a good base medium, such as T6VA, can equal the results using conditioned medium. Different cells in contact with different types of medium show variability in the pattern of responses, highlighting the presence of false positives in coculture studie

Co-culture of 1-cell outbred mouse embryos on bovine kidney epithelial cells: effect on development, glycolytic activity, inner cell mass: trophectoderm ratios and viability

In an attempt to enhance embryo development, we have co-cultured 1-cell OF1 mouse embryos on bovine kidney epithelial (Madine-Darby bovine kidney; MDBK) cells in a complex medium called complex mouse tubal fluid (cMTF; based on the energy substrate levels found in the mouse oviduct, containing non-essential amino acids, glutamine and EDTA). To determine the quality of the blastocysts obtained, we examined several parameters: morphology, total cell numbers, inner cell mass (ICM): trophectoderm (TE) ratio, glycolytic activity and viability after transfer. A significantly lower number of blastocysts developed on MDBK cells compared with cMTF medium. cMTF blastocysts had a significantly higher glycolytic activity and a lower blastocyst cell number than those grown in co-culture, while both in-vitro groups had higher ICM: TE ratios compared with in vivo. Blastocysts grown on MDBK cells displayed an elevated ICM number compared with those grown in cMTF medium alone. However, the percentage of fetuses after transfer remained drastically low in both culture groups compared with in-vivo blastocysts. In conclusion, co-culture did not increase the number of zygotes reaching the blastocyst stage. Although co-culture blastocysts show some similarities to in-vivo embryos in cell number and glycolytic activity, no enhancement in viability was observe

Chromatin packaging and morphology in ejaculated human spermatozoa: evidence of hidden anomalies in normal spermatozoa

This study aimed to investigate the association between anomalies in sperm chromatin packaging, morphology and fertilization in patients undergoing routine in-vitro fertilization (IVF) or subzonal insemination (SUZI). Sperm chromatin packaging was assessed using chromomycin A3 (CMA3), a fluorochrome specific for guanine-cytosine rich sequences of DNA. One hundred to 150 sperm cells were assessed in 55 patients to compare sperm chromatin packaging and morphology to fertilization after IVF or SUZI. When the morphology and CMA3 fluorescence of individual spermatozoa was assessed, >75% of the macrocephalic sperm fluoresced in all patients. In contrast a mean of 37% of the spermatozoa with normal morphology fluoresced in IVF patients compared with 58% of the normal spermatozoa in male factor patients treated by SUZI. SUZI patients displaying a high fluorescence (>70%) in their spermatozoa also had a significantly lower fertilization rate. Lower packaging quality in morphologically normal spermatozoa may represent a major limiting factor in the fertilizing ability of male factor patients. This study confirms that a high percentage of CMA3 positivity is present in certain forms of male factor infertility and that such a test may be used to distinguish separate populations in morphologically normal spermatozo

Intrauterine insemination: evaluation of the results according to the woman's age, sperm quality, total sperm count per insemination and life table analysis

We report on 332 infertile couples who underwent 1115 cycles of intrauterine insemination (IUI) with washed husband's semen. The indication for IUI was an abnormal post-coital test due to either a male or cervical infertility factor. The mean number of IUI cycles per patient was 3.4, the overall pregnancy rate 18, 7%, and the pregnancy rate per cycle 5.6%. The cumulative pregnancy rate calculated by life table analysis showed that 16.0% of pregnancies occurred in the first three treatment cycles, while the cumulative pregnancy rate was 26.9% by the sixth cycle. The outcome of the therapy was adversely affected if the woman's age was >39 years and/or total motile sperm count per insemination was <1X106. No pregnancy occurred in women older than 44 years or in cases with a total motile sperm count before semen preparation of <1X10

Sperm selection in natural conception:what can we learn from Mother Nature to improve assisted reproduction outcomes?

BACKGROUND: In natural conception only a few sperm cells reach the ampulla or the site of fertilization. This population is a selected group of cells since only motile cells can pass through cervical mucus and gain initial entry into the female reproductive tract. In animals, some studies indicate that the sperm selected by the reproductive tract and recovered from the uterus and the oviducts have higher fertilization rates but this is not a universal finding. Some species show less discrimination in sperm selection and abnormal sperm do arrive at the oviduct. In contrast, assisted reproductive technologies (ART) utilize a more random sperm population. In this review we contrast the journey of the spermatozoon in vivo and in vitro and discuss this in the context of developing new sperm preparation and selection techniques for ART. METHODS: A review of the literature examining characteristics of the spermatozoa selected in vivo is compared with recent developments in in vitro selection and preparation methods. Contrasts and similarities are presented. RESULTS AND CONCLUSIONS: New technologies are being developed to aid in the diagnosis, preparation and selection of spermatozoa in ART. To date progress has been frustrating and these methods have provided variable benefits in improving outcomes after ART. It is more likely that examining the mechanisms enforced by nature will provide valuable information in regard to sperm selection and preparation techniques in vitro. Identifying the properties of those spermatozoa which do reach the oviduct will also be important for the development of more effective tests of semen quality. In this review we examine the value of sperm selection to see how much guidance for ART can be gleaned from the natural selection processes in vivo

Personalized ovarian stimulation for assisted reproductive technology : study design considerations to move from hype to added value for patients

Peer reviewedPublisher PD

Male Oxidative Stress Infertility (MOSI): Proposed Terminology and Clinical Practice Guidelines for Management of Idiopathic Male Infertility

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm’s potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause

Standards in semen examination:publishing reproducible and reliable data based on high-quality methodology

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.Peer reviewe

Facilitated glucose transporters play a crucial role throughout mouse preimplantation embryo development

The role of glucose fluctuates during preimplantation mouse embryo development, indicating that a specific interplay exists between glucose metabolism and uptake. In this study, attempts were made to characterize the role of the Na+-coupled active and the facilitated glucose transporters (GLUT) during preimplantation development by using specific glucose analogues and transport inhibitors and by examining the expression of GLUT1. One-cell outbred mouse embryos were cultured in medium M16 (5.5 mmol/l glucose), M16 without glucose (M16-G), M16-G + 2-deoxyglucose, M16-G + 3-O-methylglucose, M16 + phlorizin and M16 + phloretin and development to the blastocyst stage assessed. The absence of glucose, or the presence of 3-O-methylglucose, which is taken up but not metabolized, did not inhibit blastocyst development. 2-Deoxyglucose, which is phosphorylated but not metabolized, inhibited blastocyst development. Culture in M16 supplemented with phlorizin, an inhibitor of Na+-coupled active glucose transport did not inhibit blastocyst formation. Phloretin had no effect on the cleavage of two-cell embryos to the four-cell stage, but inhibited the morula/blastocyst transition. Both phloretin and phlorizin inhibited glucose uptake in two-cell embryos. Finally, GLUT1 expression was 10-fold less in blastocysts cultured in M16 compared to in-vivo blastocysts and those cultured in M16-G. The results show that both types of glucose transporters influence preimplantation embryo development and that the embryo has an innate ability to control the uptake of glucose by regulating the expression of GLUT