The development of a complex technology such as in vitro fertilization (IVF) requires years of experimentation, sometimes comparing several species to learn how to create the right in vitro environment for oocytes, spermatozoa and early embryos. At the same time, individual species characteristics such as gamete physiology and gamete interaction are recently evolved traits and must be analysed within the context of each species. In the last 40 years since the birth of Louise Brown, IVF techniques progressed and are now used in multiple domestic and non-domestic animal species around the world. This does not mean that the technology is completely matured or satisfactory; a number of problems remain to be solved and several procedures still need to be optimized. The development of IVF in cattle is particularly interesting since agriculture practices permitted the commercial development of the procedure and it is now used at a scale comparable to human IVF (millions of newborns). The genomic selection of young animals or even embryos combined with sexing and freezing technologies is driving a new era of IVF in the dairy sector. The time has come for a retrospective analysis of the success and pitfalls of the last 40 years of bovine IVF and for the description of the challenges to overcome in the years to come.
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Bo Zheng, Jun Yu, Yueshuai Guo, Tingting Gao, Cong Shen, Xi Zhang, Hong Li and Xiaoyan Huang
The cellular nucleic acid-binding protein (CNBP), also known as zinc finger protein 9, is a highly conserved zinc finger protein that is strikingly conserved among vertebrates. Data collected from lower vertebrates showed that CNBP is expressed at high levels and distributed in the testes during spermatogenesis. However, the location and function of CNBP in mammalian testes are not well known. Here, by neonatal mouse testis culture and spermatogonial stem cells (SSC) culture methods, we studied the effect of CNBP knockdown on neonatal testicular development. Our results revealed that CNBP was mainly located in the early germ cells and Sertoli cells. Knockdown of CNBP using morpholino in neonatal testis culture caused disruption of seminiferous tubules, mislocation of Sertoli cells and loss of germ cells, which were associated with the aberrant Wnt/β-catenin pathway activation. However, knockdown of CNBP in SSC culture did not affect the survival of germ cells. In conclusion, our study suggests that CNBP could maintain testicular development by inhibiting the Wnt/β-catenin pathway, particularly by influencing Sertoli cells.
Jia Hao, Wang Yao, W B Ryan Harris, Joy Y Vink, Kristin M Myers and Eve Donnelly
The cervix shortens and softens as its collagen microstructure remodels in preparation for birth. Altered cervical tissue collagen microstructure can contribute to a mechanically weak cervix and premature cervical dilation and delivery. To investigate the local microstructural changes associated with anatomic location and pregnancy, we used second-harmonic generation microscopy to quantify the orientation and spatial distribution of collagen throughout cervical tissue from 4 pregnant and 14 non-pregnant women. Across patients, the alignment and concentration of collagen within the cervix was more variable near the internal os and less variable near the external os. Across anatomic locations, the spatial distribution of collagen within a radial zone adjacent to the inner canal of the cervix was more homogeneous than that of a region comprising the middle and outer radial zones. Two regions with different collagen distribution characteristics were found. The anterior and posterior sections in the outer radial zone were characterized by greater spatial heterogeneity of collagen than that of the rest of the sections. Our findings suggest that the microstructural alignment and distribution of collagen varies with anatomic location within the human cervix. These observed differences in collagen microstructural alignment may reflect local anatomic differences in cervical mechanical loading and function. Our study deepens the understanding of specific microstructural cervical changes in pregnancy and informs investigations of potential mechanisms for normal and premature cervical remodeling.
Darren K Griffin and Cagri Ogur
Designed to minimize chances of transferring genetically abnormal embryos, preimplantation genetic diagnosis (PGD) involves in vitro fertilization (IVF), embryo biopsy, diagnosis and selective embryo transfer. Preimplantation genetic testing for aneuploidy (PGT-A) aims to avoid miscarriage and live born trisomic offspring and to improve IVF success. Diagnostic approaches include fluorescence in situ hybridization (FISH) and more contemporary comprehensive chromosome screening (CCS) including array comparative genomic hybridization (aCGH), quantitative polymerase chain reaction (PCR), next-generation sequencing (NGS) and karyomapping. NGS has an improved dynamic range, and karyomapping can detect chromosomal and monogenic disorders simultaneously. Mosaicism (commonplace in human embryos) can arise by several mechanisms; those arising initially meiotically (but with a subsequent post-zygotic ‘trisomy rescue’ event) usually lead to adverse outcomes, whereas the extent to which mosaics that are initially chromosomally normal (but then arise purely post-zygotically) can lead to unaffected live births is uncertain. Polar body (PB) biopsy is the least common sampling method, having drawbacks including cost and inability to detect any paternal contribution. Historically, cleavage-stage (blastomere) biopsy has been the most popular; however, higher abnormality levels, mosaicism and potential for embryo damage have led to it being superseded by blastocyst (trophectoderm – TE) biopsy, which provides more cells for analysis. Improved biopsy, diagnosis and freeze-all strategies collectively have the potential to revolutionize PGT-A, and there is increasing evidence of their combined efficacy. Nonetheless, PGT-A continues to attract criticism, prompting questions of when we consider the evidence base sufficient to justify routine PGT-A? Basic biological research is essential to address unanswered questions concerning the chromosome complement of human embryos, and we thus entreat companies, governments and charities to fund more. This will benefit both IVF patients and prospective parents at risk of aneuploid offspring following natural conception. The aim of this review is to appraise the ‘state of the art’ in terms of PGT-A, including the controversial areas, and to suggest a practical ‘way forward’ in terms of future diagnosis and applied research.
Alan H Handyside
The first pregnancies and live births following in vitro fertilisation (IVF) and preimplantation genetic testing (PGT), formerly known as preimplantation genetic diagnosis, were reported in 1990, almost 30 years ago, in several couples at risk of X-linked inherited conditions, which typically only affect boys inheriting the X chromosome with the affected gene from their carrier mothers. At that time, it was only possible to identify the sex of the embryo by amplifying a Y-linked repeat sequence in single cells biopsied at cleavage stages and avoid the transfer of males, half of which would be affected. The extensive publicity surrounding these cases and the perceived risk of using IVF and PGT for desirable characteristics not related to health, such as sex selection, led to the epithet of ‘designer babies’ which continues to resonate to this day. Here, I briefly reflect on how the technology of PGT has evolved over the decades and whether it deserves this reputation. With efficient methods for whole genome amplification and the genomic revolution, we now have highly accurate universal tests that combine marker-based diagnosis of almost any monogenic disorder with the detection of aneuploidy. PGT is now clinically well established and is likely to remain a valuable alternative for couples at risk of having affected children.
C L O’Neill, S Chow, Z Rosenwaks and G D Palermo
The first conception outside of the human body that led to the birth of Louise Brown was a tremendous accomplishment, which opened the door to the utilization of assisted reproductive techniques globally. This brought the understanding that accomplishing life in a dish required several steps, the most obvious being the timing and characteristics of fertilization. It soon became obvious in the 1980s that the most disappointing phenomenon was unexpected and complete fertilization failure. Among the approaches that were attempted to treat male factor infertility, ICSI surfaced as the technique that brought the ratio of the gametes to 1:1 and was also able to grant consistent fertilization and a higher pregnancy rate. ICSI has now been implemented for a quarter of a century, proving itself as the ultimate technique utilizing ejaculated spermatozoa independent of the semen parameters and is the sole insemination method to be used with surgically retrieved spermatozoa. There are currently various indications for ICSI that are widely adopted, rendering it the most popular insemination method worldwide. The reliability of ICSI ensures its employment in upcoming techniques involving in vitro spermatogenesis and neogametogenesis.
Benjamin Fisch and Ronit Abir
Anti-cancer therapy, particularly chemotherapy, damages ovarian follicles and promotes ovarian failure. The only pharmacological means for protecting the ovaries from chemotherapy-induced injury is gonadotrophin-releasing hormone agonist, but its efficiency remains controversial; ovarian transposition is used to shield the ovary from radiation when indicated. Until the late 1990s, the only option for fertility preservation and restoration in women with cancer was embryo cryopreservation. The development of other assisted reproductive technologies such as mature oocyte cryopreservation and in vitro maturation of oocytes has contributed to fertility preservation. Treatment regimens to obtain mature oocytes/embryos have been modified to overcome various limitations of conventional ovarian stimulation protocols. In the last decades, several centres have begun cryopreserving ovarian samples containing primordial follicles from young patients before anti-cancer therapy. The first live birth following implantation of cryopreserved-thawed ovarian tissue was reported in 2004; since then, the number has risen to more than 130. Nowadays, ovarian tissue cryopreservation can be combined with in vitro maturation and vitrification of oocytes. The use of cryopreserved oocytes eliminates the risk posed by ovarian implantation of reseeding the cancer. Novel methods for enhancing follicular survival after implantation are presently being studied. In addition, researchers are currently investigating agents for ovarian protection. It is expected that the risk of reimplantation of malignant cells with ovarian grafts will be overcome with the putative development of an artificial ovary and an efficient follicle class- and species-dependent in vitro system for culturing primordial follicles.
Anamaria C Herta, Francesca Lolicato and Johan E J Smitz
The currently available assisted reproduction techniques for fertility preservation (i.e. in vitro maturation (IVM) and in vitro fertilization) are insufficient as stand-alone procedures as only few reproductive cells can be conserved with these techniques. Oocytes in primordial follicles are well suited to survive the cryopreservation procedure and of use as valuable starting material for fertilization, on the condition that these could be grown up to fully matured oocytes. Our understanding of the biological mechanisms directing primordial follicle activation has increased over the last years and this knowledge has paved the way toward clinical applications. New multistep in vitro systems are making use of purified precursor cells and extracellular matrix components and by applying bio-printing technologies, an adequate follicular niche can be built. IVM of human oocytes is clinically applied in patients with polycystic ovary/polycystic ovary syndrome; related knowhow could become useful for fertility preservation and for patients with maturation failure and follicle-stimulating hormone resistance. The expectations from the research on human ovarian tissue and immature oocytes cultures, in combination with the improved vitrification methods, are high as these technologies can offer realistic potential for fertility preservation.
Jinbi Zhang, Yang Liu, Wang Yao, Qifa Li, Honglin Liu and Zengxiang Pan
In mammals, more than 99% of ovarian follicles undergo a degenerative process known as atresia. The molecular events involved in atresia initiation remain incompletely understood. The objective of this study was to analyze differential gene expression profiles of medium antral ovarian follicles during early atresia in pig. The transcriptome evaluation was performed on cDNA microarrays using healthy and early atretic follicle samples and was validated by quantitative PCR. Annotation analysis applying current database (Sus scrofa 11.1) revealed 450 significantly differential expressed genes between healthy and early atretic follicles. Among them, 142 were significantly upregulated in early atretic with respect to healthy group and 308 were downregulated. Similar expression trends were observed between microarray data and quantitative RT-PCR confirmation, which indicated the reliability of the microarray analysis. Further analysis of the differential expressed genes revealed the most significantly affected biological functions during early atresia including blood vessel development, regulation of DNA-templated transcription in response to stress and negative regulation of cell adhesion. The pathway and interaction analysis suggested that atresia initiation associates with (1) a crosstalk of cell apoptosis, autophagy and ferroptosis rather than change of typical apoptosis markers, (2) dramatic shift of steroidogenic enzymes, (3) deficient glutathione metabolism and (4) vascular degeneration. The novel gene candidates and pathways identified in the current study will lead to a comprehensive view of the molecular regulation of ovarian follicular atresia and a new understanding of atresia initiation.