C.L. Bormann

Freeze-Dried Sperm Fertilization Leads to Full-Term Development in Rabbits

Abstract To date, the laboratory mouse is the only mammal in which freeze-dried spermatozoa have been shown to support full-term development after microinjection into oocytes. Because spermatozoa in mice, unlike in most other mammals, do not contribute centrosomes to zygotes, it is still unknown whether freeze-dried spermatozoa in other mammals are fertile. Rabbit sperm was selected as a model because of its similarity to human sperm (considering the centrosome inheritance pattern). Freeze- drying induces rabbit spermatozoa to undergo dramatic changes, such as immobilization, membrane breaking, and tail fragmentation. Even when considered to be “dead” in the conventional sense, rabbit spermatozoa freeze-dried and stored at ambient temperature for more than 2 yr still have capability comparable to that of fresh spermatozoa to support preimplantation development after injection into oocytes followed by activation. A rabbit kit derived from a freeze-dried spermatozoon was born after transferring 230 sperm-injected oocytes into eight recipients. The results suggest that freeze-drying could be applied to preserve the spermatozoa from most other species, including human. The present study also raises the question of whether rabbit sperm centrosomes survive freeze-drying or are not essential for embryonic development.

Dynamic microfunnel culture enhances mouse embryo development and pregnancy rates

BACKGROUND Despite advances in in vitro manipulation of preimplantation embryos, there is still a reduction in the quality of embryos produced leading to lower pregnancy rates compared with embryos produced in vivo. We hypothesized that a dynamic microfunnel embryo culture system would enhance outcomes by better mimicking the fluid-mechanical and biochemical stimulation embryos experience in vivo from ciliary currents and oviductal contractions. METHODS AND RESULTS Mouse embryos were cultured in microdrop-static control, microfunnel-static control or microfunnel-dynamic conditions with microfluidics. All groups tested had greater than 90% total blastocyst development from zygotes after 96 h culture. Blastocyst developmental stage was significantly enhanced (P < 0.01) under dynamic microfunnel culture conditions as evidenced by an increased percentage of hatching or hatched blastocysts (Microdrop-control 31%; Microfunnel-control 23%; Microfunnel-pulsatile 71%) and significantly higher (P < 0.01) average number of cells per blastocyst (Microdrop-control 67 +/- 3; Microfunnel-control 60 +/- 3; Microfunnel-pulsatile 109 +/- 5). Blastocyst cell numbers in dynamic microfunnel cultures (109 +/- 5) more closely matched numbers obtained from in vivo grown blastocysts (144 +/- 9). Importantly, dynamic microfunnel culture significantly improved embryo implantation and ongoing pregnancy rates over static culture to levels approaching that of in utero derived preimplantation embryos. CONCLUSIONS The improved pregnancy outcomes along with the simple and user-friendly design of the microfluidic/microfunnel system has potential to alleviate many inefficiencies in embryo production for biomedical research, genetic gain in domestic species and assisted reproductive technologies in humans.

Development of goat embryos after in vitro fertilization and parthenogenetic activation by different methods

Effective activation protocols that can be used during nuclear transfer investigations in goats need to be developed. We compared the development of IVF goat embryos with those of nonfertilized parthogenetically developing oocytes activated by treatment with either ionomycin or ethanol, both followed by immediate exposure to 6-diethylaminopurine (6-DMAP). Cumulus oocyte complexes (COCs) recovered from abattoir goat ovaries were either matured in a conventional laboratory incubator or placed in pre-equilibrated maturation medium and shipped overnight in a battery-operated dry incubator to another laboratory. Mature COCs were allocated randomly to one of three treatment groups. Group 1 oocytes (n=169 shipped, n=253 not shipped) were fertilized in vitro at 24 h postmaturation (hpm). The remaining COCs were activated at 28 hpm in either ionomycin (Group 2: n=362 shipped, n=202 not shipped), or ethanol (Group 3: n=263 shipped, n=249 not shipped). Activated oocytes were immediately incubated in 6-DMAP for 4 h. Blastocyst development was evaluated on Day 8 post-insemination/activation. Percent cleavage was comparable in shipped and nonshipped oocytes and in all treatment groups. In both shipped and nonshipped oocytes, parthenotes developing from ionomycin- and ethanol-activated oocytes had significantly greater blastocyst development (P<0.01) compared to IVF embryos (28.5 +/- 3.0, 27.4 +/- 2.8, 10.3 +/- 3.0, respectively for the nonshipped oocytes and 9.9 +/- 2.1, 10.3 +/- 2.4, 3.7 +/- 4.7 respectively for the shipped oocytes). Shipped oocytes had lower blastocyst development compared to nonshipped oocytes in the three treatment groups. The mean blastocyst cell number was not statistically different between shipped and nonshipped oocytes or among treatment groups, suggesting that all were equally viable.

Effects of semen storage and separation techniques on sperm DNA fragmentation

OBJECTIVE To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. DESIGN Controlled clinical study. SETTING An assisted reproductive technology laboratory. PATIENT(S) Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. INTERVENTION(S) One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. MAIN OUTCOME MEASURE(S) DNA fragmentation as measured by SCD. RESULT(S) There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. CONCLUSION(S) The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use.

The effect of vitamins during maturation of caprine oocytes on subsequent developmental potential in vitro.

Only a small proportion of goat oocytes selected for in vitro oocyte maturation (IVM) can successfully complete cytoplasmic maturation and support embryonic development. To produce goat blastocysts more efficiently in vitro, it is necessary to identify factors required during oocyte maturation. The objective of this study was to determine the role of vitamins during maturation of caprine oocytes in semi-defined medium on subsequent developmental capacity in vitro. Cumulus oocyte complexes (COCs) collected from a local abattoir were matured in synthetic oviductal fluid (SOF) medium supplemented with BSA, LH, FSH, and EGF in the presence or absence of MEM vitamins for 24 h. The COCs were co-incubated with frozen-thawed sperm in Bracket and Oliphant fertilization medium for 18-22 h. Embryos were cultured in G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 72 h. Addition of vitamins significantly increased (P<0.05) overall blastocyst development (16.4+/-1.2% versus 12.3+/-1.1%), and tended to increase (P<0.06) the percentage of cleaved embryos (61.4+/-3.0% versus 52.7+/-2.6%). Addition of MEM vitamins to SOF maturation medium significantly increased (P<0.05) mean blastocyst cell number compared with control medium (107.7+/-6.0 versus 85.1+/-6.3). Hatched blastocysts tended to have increased (P<0.06) cell numbers in the vitamin-treated group (150.5+/-8.4 versus 123.4+/-8.8). These results suggest that addition of vitamins during oocyte maturation is beneficial for subsequent blastocyst development and viability.

Development and viability of in vitro derived porcine blastocysts cultured in NCSU23 and G1.2/G2.2 sequential medium.

Porcine embryo development in vitro is relatively inefficient compared to other domestic species. Currently, a single culture medium (NCSU23) is the standard for porcine in vitro systems. However, the G1.2/G2.2 sequential culture system has been beneficial for embryo development in other species. The objective of this study was to compare porcine preimplantation embryo development in vitro and subsequent blastocyst viability and metabolic activity using NCSU23 and G1.2/G2.2 culture media. Oocytes were matured in defined TCM199 base medium for 45 to 47 h and fertilized in mTBM for 4 h. Embryos were cultured in either NCSU23 for 146 h or G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 74 h. Blastocyst substrate use was measured using a modification of the hanging drop technique. Culture in NCSU23 resulted in a higher percentage (P < 0.05) of embryo cleavage (74.0%) and blastocyst development (14.6%) than culture in G1.2/G2.2 (67.8% and 7.8%, respectively). Both NCSU23 and G1.2/G2.2 produced blastocysts with similar mean cell numbers (51.5 +/- 4.3 and 47.1 +/- 4.3, respectively), similar glucose use (10.81 +/- 1.39 and 10.12 +/- 1.72 pmol/embryo/3 h, respectively) and pyruvate use (1.08 +/- 0.056 and 0.88 +/- 0.048 pmol/embryo/3 h, respectively). These data indicate that a sequential culture system can support porcine embryo development in vitro without compromising embryo viability. However, the G1.2/G2.2 system was not as effective as NCSU23 in supporting blastocyst development. Sequential media should be formulated specifically for porcine embryos to improve embryonic cleavage and blastocyst development.

An automated smartphone-based diagnostic assay for point-of-care semen analysis

This work demonstrates that a low-cost smartphone accessory can be used for home-based male infertility screening. Sperm samples phoning in Although male infertility is as common as female infertility, it often goes undiagnosed because of socioeconomic factors such as stigma, high cost of testing, and availability of laboratory facilities. To facilitate the necessary testing, Kanakasabapathy et al. have designed a smartphone-based assay that can be performed at home or in a remote clinic without access to laboratory equipment. The assay uses an inexpensive device that attaches directly to a phone and is operated through a smartphone application. The accuracy of this approach was very similar to that of computer-assisted laboratory analysis, even when it was performed by untrained users with no clinical background, demonstrating its potential for use at home and in low-resource settings. Male infertility affects up to 12% of the world’s male population and is linked to various environmental and medical conditions. Manual microscope-based testing and computer-assisted semen analysis (CASA) are the current standard methods to diagnose male infertility; however, these methods are labor-intensive, expensive, and laboratory-based. Cultural and socially dominated stigma against male infertility testing hinders a large number of men from getting tested for infertility, especially in resource-limited African countries. We describe the development and clinical testing of an automated smartphone-based semen analyzer designed for quantitative measurement of sperm concentration and motility for point-of-care male infertility screening. Using a total of 350 clinical semen specimens at a fertility clinic, we have shown that our assay can analyze an unwashed, unprocessed liquefied semen sample with <5-s mean processing time and provide the user a semen quality evaluation based on the World Health Organization (WHO) guidelines with ~98% accuracy. The work suggests that the integration of microfluidics, optical sensing accessories, and advances in consumer electronics, particularly smartphone capabilities, can make remote semen quality testing accessible to people in both developed and developing countries who have access to smartphones.

Prenatal testosterone and dihydrotestosterone exposure disrupts ovine testicular development

Androgens play important roles during the first trimester of intrauterine life, coinciding with genital tract differentiation, during virilization and maintenance of secondary male characteristics, and during initiation of spermatogenesis. Little is known about the impact of inappropriate exposure to excess androgens during fetal development on male sexual maturation and reproduction. The objectives of this study were to determine the effects of prenatal 5α-dihydrotestosterone (DHT) and testosterone treatment during ovine sexual differentiation on post-pubertal testicular formation and subsequent potential for fertility as assessed by epididymal sperm characteristics. Rams prenatally treated with testosterone exhibited increased testicular weight relative to age-matched controls and prenatal DHT-treated rams (P<0.05), as well as elevated total and free testosterone concentrations compared with DHT-treated rams (P=0.07 and P<0.05 respectively). The percentage of progressively motile sperm from the epididymis was significantly reduced in prenatal DHT-treated but not testosterone-treated rams compared with control rams (P<0.05). The testosterone-treated rams had a greater number of germ cell layers than DHT-treated rams, but comparable to the controls. Prenatal testosterone-treated rams had significantly larger seminiferous tubule diameter and lumen diameter compared with prenatal DHT-treated (P<0.05). Significantly, more prenatal DHT- and testosterone-treated rams (P<0.05) had occluded tubule lumen than control rams. Findings from this study demonstrate that exposure to excess testosterone/DHT during male fetal sexual differentiation have differential effects on post-pubertal testicular size, seminiferous tubule size and function, sperm motility, and testosterone concentrations.

Microfluidics for Gametes, Embryos, and Embryonic Stem Cells

Microfluidics is a young but established field that holds significant potential for scientific discovery. The utility of microfluidics can improve our knowledge of basic biology as well as expand our understanding in specialized areas such as assisted reproduction and stem cell developmental biology. This review describes the technology of microfluidics and discusses applications within assisted reproduction technology and embryonic stem cell growth and directed differentiation. Development of an integrated microfluidic platform for assisted reproduction, which can manipulate gametes, embryos, embryonic stem cells, their culture environment, and incorporate biomarker analysis, could have a dramatic impact on the basic understanding of embryo/embryonic stem cell development, as well as provide significant improvements in current technologies used to treat infertility, preserve fertility, and derive therapeutic cells from stem cells.

Induction of chemokines and prostaglandin synthesis pathways in luteinized human granulosa cells: potential role of luteotropin withdrawal and prostaglandin F2α in regression of the human corpus luteum.

Our objective was to determine the effects of prostaglandin F2α (PGF2α) and withdrawal of luteotropic stimulants (forskolin or hCG) on expression of chemokines and prostaglandin-endoperoxide synthase 2 (PTGS2) in luteinized human granulosa cells. Human granulosa cells were collected from 12 women undergoing oocyte retrieval and were luteinized in vitro with forskolin or hCG. In first experiment, granulosa-lutein cells were treated with PGF2α, the primary luteolytic hormone in most species. In second experiment, granulosa cells that had been luteinized for 8 d had luteotropins withdrawn for 1, 2, or 3 d. Treatment with PGF2α induced mRNA for chemokine (c-x-c motif) ligand 2 (CXCL2) and CXC ligand 8 (CXCL8; also known as interleukin-8) in granulosa cells luteinized for 8 d but not in cells that were only luteinized for 2 d. Similarly, luteinization of human granulosa cells for 8 d with forskolin or hCG followed by withdrawal of luteotropic stimulants, not only decreased P4 production, but also increased mRNA concentrations for CXCL8, CXCL-2 (after forskolin withdrawal), and PTGS2. These results provide evidence for two key steps in differentiation of luteolytic capability in human granulosa cells. During 8 d of luteinization, granulosa cells acquire the ability to respond to luteolytic factors, such as PGF2α, with induction of genes involved in immune function and PG synthesis. Finally, a decline in luteotropic stimuli triggers similar pathways leading to induction of PTGS2 and possibly intraluteal PGF2α production, chemokine expression, leukocyte infiltration and activation, and ultimately luteal regression.