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“Functional Primordial Germ Cell-like Cells from Pluripotent Stem Cells in Rats”, Oikawa et al 2022

2022-oikawa.pdf: “Functional primordial germ cell-like cells from pluripotent stem cells in rats”⁠, Mami Oikawa, Hisato Kobayashi, Makoto Sanbo, Naoaki Mizuno, Kenyu Iwatsuki, Tomoya Takashima, Keiko Yamauchi et al (2022-04-11; similar):

Generating functional rate gametes: In the past decade, methods have been developed to generate germ cells from pluripotent stem cells for studies of development and in vitro gametogenesis⁠. However, offspring from in vitro-derived germ cells has only been achieved in mice. Oikawa et al 2022 extend this work beyond mice to a second rodent species, the rat, a leading animal model for biomedical research with many physiological similarities to humans. A stepwise protocol allows for the production of fetal stage rat germ cells that can produce viable offspring upon maturation in the testis and injection of the sperm into unfertilized oocytes. This system will allow comparative studies and enable broader execution and analysis of in vitro gametogenesis.


The in vitro generation of germ cells from pluripotent stem cells (PSCs) can have a substantial effect on future reproductive medicine and animal breeding. A decade ago, in vitro gametogenesis was established in the mouse. However, induction of primordial germ cell-like cells (PGCLCs) to produce gametes has not been achieved in any other species.

Here, we demonstrate the induction of functional PGCLCs from rat PSCs. We show that epiblast-like cells in floating aggregates form rat PGCLCs.

The gonadal somatic cells support maturation and epigenetic reprogramming of the PGCLCs. When rat PGCLCs are transplanted into the seminiferous tubules of germline-less rats, functional spermatids—that is, those capable of siring viable offspring—are generated.

Insights from our rat model will elucidate conserved and divergent mechanisms essential for the broad applicability of in vitro gametogenesis.

“Haploidy in Somatic Cells Is Induced by Mature Oocytes in Mice”, Lee et al 2022

“Haploidy in somatic cells is induced by mature oocytes in mice”⁠, Yeonmi Lee, Aysha Trout, Nuria Marti-Gutierrez, Seoon Kang, Philip Xie, Aleksei Mikhalchenko, Bitnara Kim et al (2022-01-25; similar):

Haploidy is naturally observed in gametes⁠; however, attempts of experimentally inducing haploidy in somatic cells have not been successful.

Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0⁠/​G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids⁠. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos.

Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.

“Generation of Ovarian Follicles from Mouse Pluripotent Stem Cells”, Yoshino et al 2021

2021-yoshino.pdf: “Generation of ovarian follicles from mouse pluripotent stem cells”⁠, Takashi Yoshino, Takahiro Suzuki, Go Nagamatsu, Haruka Yabukami, Mika Ikegaya, Mami Kishima, Haruka Kita et al (2021-07-16; backlinks; similar):

Oocytes mature in a specialized fluid-filled sac, the ovarian follicle, which provides signals needed for meiosis and germ cell growth. Methods have been developed to generate functional oocytes from pluripotent stem cell-derived primordial germ cell-like cells (PGCLCs) when placed in culture with embryonic ovarian somatic cells. In this study, we developed culture conditions to recreate the stepwise differentiation process from pluripotent cells to fetal ovarian somatic cell-like cells (FOSLCs). When FOSLCs were aggregated with PGCLCs derived from mouse embryonic stem cells, the PGCLCs entered meiosis to generate functional oocytes capable of fertilization and development to live offspring. Generating functional mouse oocytes in a reconstituted ovarian environment provides a method for in vitro oocyte production and follicle generation for a better understanding of mammalian reproduction.

“Artificially Produced Gametes in Mice, Humans and Other Species”, Hayashi et al 2021

2021-hayashi.pdf: “Artificially produced gametes in mice, humans and other species”⁠, Katsuhiko Hayashi, Cesare Galli, Sebastian Diecke, Thomas B. Hildebrandt (2021-01-08; backlinks; similar):

The production of gametes from pluripotent stem cells in culture, also known as invitro gametogenesis, will make an important contribution to reproductive biology and regenerative medicine, both as an unique tool for understanding germ cell development and as an alternative source of gametes for reproduction. Invitro gametogenesis was developed using mouse pluripotent stem cells but is increasingly being applied in other mammalian species, including humans. In principle, the entire process of germ cell development is nearly reconstitutable in culture using mouse pluripotent stem cells, although the fidelity of differentiation processes and the quality of resultant gametes remain to be refined. The methodology in the mouse system is only partially applicable to other species, and thus it must be optimized for each species. In this review, we update the current status of invitro gametogenesis in mice, humans and other animals, and discuss challenges for further development of this technology.

“Eight Proteins Turn Mouse Stem Cells into Egglike Cells: The Identification of the Transcription Factors That Elicit Oocyte Growth Will Aid Reproductive Biology Research and Might Help Women With Fertility Issues, Scientists Say”, Yeager 2020

“Eight Proteins Turn Mouse Stem Cells into Egglike Cells: The identification of the transcription factors that elicit oocyte growth will aid reproductive biology research and might help women with fertility issues, scientists say”⁠, Ashley Yeager (2020-12-16; ; similar):

“This demonstrates that you can go directly from stem cells to oocytes. I think that is exciting”, Petra Hajkova, a developmental epigeneticist at Imperial College London who was not involved in the study, tells The Scientist. The work, she notes, will help researchers explore the basic biology of oocyte development. In the future, says study coauthor Nobuhiko Hamazaki of Kyushu University, the research could aid in cloning endangered animals or helping women with mitochondrial diseases to have healthy children.

…“It’s believed that oocytes develop from germ cells, but we could make oocytes from non-germ cells”, he explains. “At first, I was so surprised that I could not believe my results, so I repeated the experiment again and again and when I got the same results, I was finally convinced.”…“I was initially in complete disbelief to see mouse stem cells so quickly and easily take the form of oocytes based on introducing just a handful of factors, but repeated experiments proved it was true”, said Nobuhiko Hamazaki, PhD, first author on the study reporting the results and assistant professor at Kyushu University at the time of the research. “To find that eight transcription factors could lead to such big changes was quite astonishing.”

…Richard Schultz, a cell biologist at the University of California, Davis, who was not involved in the study, says the work to identify the core set of transcription factors that can drive embryonic stem cells into a state where they look like oocytes is impressive. But the egglike cells don’t undergo meiosis, so they are not functional. “It’s a big step, but only 95% there. We haven’t gotten 100% there” to understanding the factors essential for maturation of germline egg cells to oocytes and then to viable eggs with half their chromosomes. Despite not working out the pathway to meiosis, the work “enabled us to produce a large number of oocytes. We believe that this technology can accelerate basic biological research on oocytes, which are still one of the most mysterious cell types”, Hamazaki says. He explains that the work could improve animal cloning because of the vast number of oocytes produced by the team’s technique…“Cytoplasm from oocytes is an invaluable resource in reproductive biology and medicine, and this method could provide a novel tool for producing large amounts of it without any invasive procedures”, commented Hayashi. “While the processes could still be much more complex for humans, these initial results in mice are very promising.”

“Functional Oocytes Derived from Granulosa Cells”, Tian et al 2019

“Functional Oocytes Derived from Granulosa Cells”⁠, Chenglei Tian, Linlin Liu, Xiaoying Ye, Haifeng Fu, Xiaoyan Sheng, Lingling Wang, Huasong Wang, Dai Heng et al (2019-12-24; backlinks; similar):

  • Granulosa cells can be reprogrammed to form oocytes by chemical reprogramming
  • Rock inhibition and crotonic acid facilitate the chemical induction of gPSCs from GCs
  • PGCLCs derived from gPSCs exhibit longer telomeres and high genomic stability

The generation of genomically stable and functional oocytes has great potential for preserving fertility and restoring ovarian function. It remains elusive whether functional oocytes can be generated from adult female somatic cells through reprogramming to germline-competent pluripotent stem cells (gPSCs) by chemical treatment alone. Here, we show that somatic granulosa cells isolated from adult mouse ovaries can be robustly induced to generate gPSCs by a purely chemical approach, with additional Rock inhibition and critical reprogramming facilitated by crotonic sodium or acid. These gPSCs acquired high germline competency and could consistently be directed to differentiate into primordial-germ-cell-like cells and form functional oocytes that produce fertile mice. Moreover, gPSCs promoted by crotonylation and the derived germ cells exhibited longer telomeres and high genomic stability like PGCs in vivo, providing additional evidence supporting the safety and effectiveness of chemical induction, which is particularly important for germ cells in genetic inheritance.

[Keywords: chemical reprogramming, pluripotent stem cell, oocyte, granulosa cell]

“Mouse Pups Born from Eggs Derived from the Granulosa Cells That Surround Oocytes”, Press 2019

“Mouse pups born from eggs derived from the granulosa cells that surround oocytes”⁠, Cell Press (2019-12-24; similar):

Ovarian follicles are the basic functional unit of the ovary and consist of an oocyte, the immature egg, which is surrounded by granulosa cells. Besides being crucial to the development of follicles, studies have shown that granulosa cells possess plasticity that shows stem cell-like properties.

“The thing about in vitro fertilization is that they only use the oocyte for the procedure”, says senior author Lin Liu, of the College of Life Sciences at Nankai University. “After the egg retrieval, the granulosa cells in the follicle are discarded. It got us thinking, what if we can utilize these granulosa cells? Since every egg has thousands of granulosa cells surrounding it, if we can induce them into pluripotent cells and turn those cells into oocytes, aren’t we killing two birds with one stone?”

Granulosa cells tend to undergo cell death and differentiation once removed from the follicles. Liu and his team including Ph.D. students Chenglei Tian and Haifeng Fu developed a chemical “cocktail” with Rock inhibitor and crotonic acid for creating chemically induced pluripotent stem cells (CiPSCs) from granulosa cells. The research team introduced Rock inhibitor to prevent cell death and promote proliferation. In combination with other important small chemicals, crotonic acid facilitates the induction of granulosa cells into germline-competent pluripotent stem cells that exhibit pluripotency similar to embryonic stem cells.

“Controlled Modelling of Human Epiblast and Amnion Development Using Stem Cells”, Zheng et al 2019

2019-zheng.pdf: “Controlled modelling of human epiblast and amnion development using stem cells”⁠, Yi Zheng, Xufeng Xue, Yue Shao, Sicong Wang, Sajedeh Nasr Esfahani, Zida Li, Jonathon M. Muncie, Johnathon N. Lakins et al (2019-09-11; backlinks; similar):

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.

“Gametes from Stem Cells: Status and Applications in Animal Reproduction”, Goszczynski et al 2019

2019-goszczynski.pdf: “Gametes from stem cells: Status and applications in animal reproduction”⁠, Daniel E. Goszczynski, Anna C. Denicol, Pablo J. Ross (2019-07-03; backlinks; similar):

In vitro gamete differentiation could revolutionize animal production by decreasing generation intervals, increasing the number of gametes per animal and facilitating the dissemination of elite genetics. In addition, it could help to develop new strategies for the conservation of endangered species. The recent in vitro reconstitution of germ cell development in mice has inspired researchers to invest their best efforts into reproducing this achievement in livestock species.

With this goal in mind, multiple differentiation approaches and cell sources have been evaluated. The degree of success in these evaluations varies according to the species and the stage of development studied, but, in general, partially positive results have been obtained. Evidence suggests that although functional gametes with true reproductive potential are still to be obtained, it is a matter of time before this goal is achieved. [Previously: “In vitro breeding: application of embryonic stem cells to animal production”⁠, Goszczynski et al 2018]

“Generation of Human Oogonia from Induced Pluripotent Stem Cells in Vitro”, Yamashiro et al 2018

2018-yamashiro.pdf: “Generation of human oogonia from induced pluripotent stem cells in vitro”⁠, Chika Yamashiro, Kotaro Sasaki, Yukihiro Yabuta, Yoji Kojima, Tomonori Nakamura, Ikuhiro Okamoto, Shihori Yokobayashi et al (2018-01-01; )

“In Vitro Gametogenesis and Reproductive Cloning: Can We Allow One While Banning the Other?”, Segers et al 2018

2018-segers.pdf: “In vitro gametogenesis and reproductive cloning: Can we allow one while banning the other?”⁠, Seppe Segers, Guido Pennings, Wybo Dondorp, Guido de Wert, Heidi Mertes (2018-01-01; )

“In Vitro Breeding: Application of Embryonic Stem Cells to Animal Production”, Goszczynski et al 2018

2018-goszczynski.pdf: “In vitro breeding: application of embryonic stem cells to animal production”⁠, Daniel E. Goszczynski, Hao Cheng, Sebastian Demyda-Peyrás, Juan F. Medrano, Jun Wu, Pablo J. Ross (2018-01-01; backlinks)

“An Extra-uterine System to Physiologically Support the Extreme Premature Lamb”, Partridge et al 2017

“An extra-uterine system to physiologically support the extreme premature lamb”⁠, Emily A. Partridge, Marcus G. Davey, Matthew A. Hornick, Patrick E. McGovern, Ali Y. Mejaddam, Jesse D. Vrecenak et al (2017-04-25; ):

In the developed world, extreme prematurity is the leading cause of neonatal mortality and morbidity due to a combination of organ immaturity and iatrogenic injury. Until now, efforts to extend gestation using extracorporeal systems have achieved limited success.

Here we report the development of a system that incorporates a pumpless oxygenator circuit connected to the fetus of a lamb via an umbilical cord interface that is maintained within a closed ‘amniotic fluid’ circuit that closely reproduces the environment of the womb.

We show that fetal lambs that are developmentally equivalent to the extreme premature human infant can be physiologically supported in this extra-uterine device for up to 4 weeks. Lambs on support maintain stable haemodynamics, have normal blood gas and oxygenation parameters and maintain [openness] of the fetal circulation. With appropriate nutritional support, lambs on the system demonstrate normal somatic growth, lung maturation and brain growth and myelination⁠.

“MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency”, Zhang et al 2016

2016-zhang.pdf: “MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency”⁠, Hui Zhang, Srimonta Gayen, Jie Xiong, Bo Zhou, Avinash K. Shanmugam, Yuqing Sun, Hacer Karatas, Liu Liu et al (2016-01-01; backlinks)

“Chapter 10: Applications of In Vitro Techniques in Plant Breeding”, Zulkarnain et al 2015

2015-zulkarnain.pdf: “Chapter 10: Applications of In Vitro Techniques in Plant Breeding”⁠, Zul Zulkarnain, Tanya Tapingkae, Acram Taji (2015-01-01; backlinks)

“In Vitro Eugenics”, Sparrow 2014

2014-sparrow.pdf: “In vitro eugenics”⁠, Robert Sparrow (2014-11; backlinks; similar):

A series of recent scientific results suggest that, in the not-too-distant future, it will be possible to create viable human gametes from human stem cells. This paper discusses the potential of this technology to make possible what I call “in vitro eugenics”: the deliberate breeding of human beings in vitro by fusing sperm and egg derived from different stem-cell lines to create an embryo and then deriving new gametes from stem cells derived from that embryo. Repeated iterations of this process would allow scientists to proceed through multiple human generations in the laboratory. In vitro eugenics might be used to study the heredity of genetic disorders and to produce cell lines of a desired character for medical applications. More controversially, it might also function as a powerful technology of ‘human enhancement’ by allowing researchers to use all the techniques of selective breeding to produce individuals with a desired genotype.

“Human in Vitro Eugenics: Close, yet Far Away”, Fonseca et al 2014

2013-dafonseca.pdf: “Human in vitro eugenics: close, yet far away”⁠, Flávio Guimarães da Fonseca, Daniel Mendes Ribeiro, Nara Pereira Carvalho, Brunello Stancioli (2014-01-01; backlinks)

“Perspectives of Germ Cell Development in Vitro in Mammals”, Hayashi & Saitou 2014

“Perspectives of germ cell development in vitro in mammals”⁠, Katsuhiko Hayashi, Mitinori Saitou (2014; backlinks; similar):

Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are able to differentiate into all cell lineages of the embryo proper, including germ cells. This pluripotent property has a huge impact on the fields of regenerative medicine, developmental biology and reproductive engineering. Establishing the germ cell lineage from ESCs/​iPSCs is the key biological subject, since it would contribute not only to dissection of the biological processes of germ cell development but also to production of unlimited numbers of functional gametes in vitro. Toward this goal, we recently established a culture system that induces functional mouse primordial germ cells (PGCs), precursors of all germ cells, from mouse ESCs/​iPSCs. The successful in vitro production of PGCs arose from the study of pluripotent cell state, the signals inducing PGCs and the technology of transplantation. However, there are many obstacles to be overcome for the robust generation of mature gametes or for application of the culture system to other species, including humans and livestock. In this review, we discuss the requirements for a culture system to generate the germ cell lineage from ESCs/​iPSCs.

“Procreative Beneficence and in Vitro Gametogenesis”, Bourne et al 2012

“Procreative beneficence and in vitro gametogenesis”⁠, Hannah Bourne, Thomas Douglas, Julian Savulescu (2012-09-01; backlinks; similar):

The Principle of Procreative Beneficence (PB) holds that when a couple plans to have a child, they have substantial moral reason to select, of the possible children they could have, the child who is most likely to experience the greatest wellbeing—that is, the most advantaged child, the child with the best chance at the best life…In this paper we wish address a different and more practical objection: the objection that parents will be heavily restricted in the number of traits that they can select, since they will have to choose among a very limited number of embryos.

Recent advances in stem cell research may provide a solution to this problem. Recent research suggests that it may become possible to derive gametes (eggs and sperm) from human stem cells in vitro, a process which we will term in vitro gametogenesis (IVG). IVG would allow the creation of stems cells from a patient’s somatic (body) cells, and these stems cells could then be used to generate a plentiful supply of eggs or sperm in the laboratory…The ability to create large numbers of eggs or sperm through IVG greatly increases our capacity to select the best child possible. Selection could occur in two ways: (1) the most genetically desirable of this massive number of gametes could be selected and then used to create an embryo, or alternatively, (2) large numbers of embryos could be produced from these gametes and then the best embryo selected. Whatever the method, the advent of IVG could allow us to select for a much larger number of traits than is currently conceivable.

…Suppose that a couple would like to select for 20 single gene traits which are carried on 20 different and unlinked autosomal loci. Suppose further that at ten of these loci, alleles contribute recessively to the desired trait…The chance of the couple having such a child would be just over 1% with traditional IVF plus selection, but would increased to over 99.99% if 10,000 embryos could be created using IVG.

…By enabling the creation of large numbers of gametes and embryos, IVG may allow the selection of traits in future children to a degree that has previously been inconceivable.

“Pluripotent Stem Cell-derived Gametes: Truth and (potential) Consequences”, Mathews et al 2009

“Pluripotent stem cell-derived gametes: truth and (potential) consequences”⁠, Debra J. H Mathews, Peter J. Donovan, John Harris, Robin Lovell-Badge, Julian Savulescu, Ruth Faden (2009; backlinks; similar):

An emerging body of data suggests that pluripotent stem cells may be able to differentiate to form eggs and sperm. We discuss the state of the science and the potential social implications and offer recommendations for addressing some of the ethical and policy issues that would be raised by the availability of stem cell-derived gametes.

“Strategies to Utilize Marker-Quantitative Trait Loci Associations”, Haley & Visscher 1998

1998-haley.pdf: “Strategies to Utilize Marker-Quantitative Trait Loci Associations”⁠, C. S. Haley, P. M. Visscher (1998; ; backlinks; similar):

Marker-assisted selection holds promise because genetic markers provide completely heritable traits that can be measured at any age in either sex and that are potentially correlated with traits of economic value. Theoretical and simulation studies show that the advantage of using marker-assisted selection can be substantial, particularly when marker information is used, because normal selection is less effective, for example, for sex-limited or carcass traits. Assessment of the available information and its most effective use is difficult, but approaches such as crossvalidation may help in this respect. Marker systems are now becoming available that allow the high density of markers required for close associations between marker loci and trait loci. Emerging technologies could allow large numbers of polymorphic sites to be identified, practically guaranteeing that markers will be available that are in complete association with any trait locus. Identifying which polymorphism out of many that is associated with any trait will remain problematic, but multiple-locus disequilibrium measures may allow performance to be associated with unique marker haplotypes⁠. This type of approach, combined with cheap and high density markers, could allow a move from selection based on a combination of “infinitesimal” effects plus individual loci to effective total genomic selection. In such an unified model, each region of the genome would be given its appropriate weight in a breeding program. However, the collection of good quality trait information will remain central to the use of these technologies for the foreseeable future.

[Keywords: markers, breeding, quantitative trait loci⁠, selection]

“Velogenetics, or the Synergistic Use of Marker Assisted Selection and Germ-line Manipulation”, Georges & Massey 1991

1991-georges.pdf: “Velogenetics, or the synergistic use of marker assisted selection and germ-line manipulation”⁠, M. Georges, J. M. Massey (1991; backlinks; similar):

Until recently, artificial selection has relied on the biometrical evaluation of individual breeding values from an animal’s own performance and from performance of its relatives. This biometrical strategy is based on relatively simple genetic premises, operating within a “black box”. Briefly, the majority of economically important traits are so-called complex or quantitative traits, meaning that the phenotype of an animal is determined by both environment and a large number of genes with individually small, additive effects. The proportion of the phenotypic variation observed in a given population that is genetic in nature is the heritability of the trait. Substantial genetic progress has been obtained using this approach. One of the powers of the biometrical approach is that it obviates the need for any detailed molecular knowledge of the underlying genes or Economic Trait Loci (ETL).

However, it is believed that the molecular identification of these BTLs should allow for an increased genetic response by affecting both time and accuracy of selection, through a procedure called Marker Assisted Selection (MAS) (1,2). Moreover, we propose to use a scheme that we call “velogenetics”, or the combined use of Marker Assisted Selection and germ-line manipulations aimed at shortening the generation interval of domestic species (especially cattle), which would allow the efficient introgression of mapped Economic Trait Loci between genetic backgrounds.

“Potential Genetic Improvement of Cattle by Fertilization of Fetal Oocytes in Vitro”, Betteridge et al 1989

1989-betteridge.pdf: “Potential genetic improvement of cattle by fertilization of fetal oocytes in vitro”⁠, K. J. Betteridge, C. Smith, R. B. Stubbings, K. P. Xu, W. A. King (1989-01-01; backlinks)

Stem cell

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Spermatogenesis

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Organoids

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Oogenesis

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Micropropagation

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Integer programming

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Gametogenesis

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Gamete § Artificial gametes

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Cryopreservation

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Constraint (mathematics)

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