Canine and Feline Endocrinology and Reproduction

Infertility is a growing global health issue with far-reaching socioeconomic implications. A downward trend in male fertility highlights the acute need for affordable and accessible diagnosis and treatment. Assisted reproductive technologies are effective in treating male infertility, but their success rate has plateaued at ∼33% per cycle. Many emerging opportunities exist for microfluidics - a mature technology in other biomedical areas - in male infertility diagnosis and treatment, and promising microfluidic approaches are under investigation for addressing male infertility. Microfluidic approaches can improve our fundamental understanding of sperm motion, and developments in microfluidic devices that use microfabrication and sperm behaviour can aid semen analysis and sperm selection. Many burgeoning possibilities exist for engineers, biologists, and clinicians to improve current practices for infertility diagnosis and treatment. The most promising avenues have the potential to improve medical practice, moving innovations from research laboratories to clinics and patients in the near future.

Microfluidics for sperm analysis and selection

A long-standing question in natural reproduction is how mammalian sperm navigate inside female reproductive tract and finally reach the egg cell, or oocyte. Recently, fluid flow was proposed as a long-range guidance cue for sperm navigation. Coitus induces fluid flow from oviduct to uterus, and sperm align themselves against the flow direction and swim upstream, a phenomenon termed rheotaxis. Whether sperm rheotaxis is a passive process dominated by fluid mechanics, or sperm actively sense and adapt to fluid flow remains controversial. Here we report the first quantitative study of sperm flagellar motion during human sperm rheotaxis and provide direct evidence indicating that sperm rheotaxis is a passive process. Experimental results show that there is no significant difference in flagellar beating amplitude and asymmetry between rheotaxis-turning sperm and those sperm swimming freely in the absence of fluid flow. Additionally, fluorescence image tracking shows no Ca(2+) influx during sperm rheotaxis turning, further suggesting there is no active signal transduction during human sperm rheotaxis.

/pdf/human-sperm-rheotaxis-a-passive-physical-process-375s35xtk6.pdf

Human sperm rheotaxis: a passive physical process.

Almost 50% of the infertility cases are due to male factors. Assisted reproductive technologies (ARTs) allow to overcome the incapacity of these patients' spermatozoa to fertilize the oocyte and produce a viable and healthy offspring, but the efficiency of the different techniques has still the potential to improve. According to the latest reports of the European Society of Human Reproduction and Embryology (ESHRE) and the Centers for Disease Control and Prevention of the United States (CDC), the percentages of deliveries per ART cycle in 2014 and 2016 were 21 and 22%, respectively. Among the reasons for this relatively low efficiency, the quality of the spermatozoa has been pointed out as critical, and the presence of high percentages of DNA-damaged spermatozoa in patients' ejaculates is possibly one of the main factors reducing the ARTs outcomes. Thus, one of the main challenges in reproductive medicine is to ensure the highest quality of the spermatozoa used in ARTs, and specifically, in terms of genetic integrity. The latest techniques for the preparation and selection of human spermatozoa are herein discussed focusing on those proven to improve one or several of the following parameters: sperm genetic integrity, fertilization capacity, embryo production, and in vitro survival, as well as pregnancy and delivery rates following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In addition, we discuss the potential of techniques developed in non-human mammals that could be further transferred to the clinic.

/pdf/novel-techniques-of-sperm-selection-for-improving-ivf-and-3y31xi6kri.pdf

Novel Techniques of Sperm Selection for Improving IVF and ICSI Outcomes.

The fields of assisted reproductive technology (ART) and in vitro fertilization (IVF) have progressed rapidly, yet still need further improvements. Microfluidic technology can incorporate various ART procedures such as embryo/gamete (sperm/oocyte) analysis, sorting, manipulation, culture, and monitoring. The introduction of paper‐based and droplet‐based microfluidics further improves the commercialization potential of this technology. The progress in 3D printing technology allows for the integration of microfluidics with tissue engineering that may revolutionize current practices in biology and medicine. This review categorizes ART methods according to continuous‐flow microfluidics, paper‐based microfluidics, droplet‐based microfluidics, and organ‐on‐a‐chip. The advances are summarized and potential opportunities in infertility diagnosis, sperm selection, sperm guidance, oocyte selection, insemination, embryo culture, embryo monitoring, and cryopreservation are identified. While some advances of continuous‐flow microfluidics for ART have already been reviewed, other microfluidic techniques are still in their early stages. It is envisioned that advances in droplet‐based microfluidics, especially digital microfluidics, will allow for more progress in human IVF, particularly single embryo transfer. Droplet‐based microfluidics may also lead to fully integrated and high‐throughput platforms for animal IVF. Recent advances in organ‐on‐a‐chip including ovary/uterus/oviduct‐on‐chip platforms hold promise for the integration of the whole human reproductive system‐on‐a‐chip for clinical applications.

Advances in Microfluidics-Based Assisted Reproductive Technology: From Sperm Sorter to Reproductive System-on-a-Chip

We study rheotaxis of bull sperm inside microchannels to characterize the effects of flow and wall shape on sperm swimming behavior. We found that a large percentage of sperm cells, 80 to 84%, exhibited positive rheotaxis (sperm cells swimming against the flow) within flow velocities of 33 to 134 μm s−1. Sperm cells were also found to reverse their swimming direction when the liquid flow direction was reversed. Time taken by sperm cells to reverse their swimming direction was inversely proportional to the flow velocity. Sperm behavior was significantly affected by the sperm position with respect to the channel wall. Sperm cells close to the channel wall moved upstream faster than sperm cells moving along the channel centerline. Shear stress, which is an indicator of velocity distribution, was found to play an important role in regulating rheotactic behavior of sperm cells. Side pockets were added to some microchannels to mimic storage sites in mucosal folds and pockets in the fallopian tube of the female reproductive system and sperm interaction with these pockets was monitored. We found that sperm cells tend to follow channel walls and enter these pockets without any chemical binding, which further confirms the wall tracking behavior of mammalian sperm cells. Our results confirm that sperm rheotaxis is a strong mechanism for guiding sperm cells towards the oocyte along the female genital tract.

Characterization of rheotaxis of bull sperm using microfluidics

Development of computer-assisted sperm analysis plugin for analyzing sperm motion in microfluidic environments using Image-J.

The aim of the present study was to study the effect of parity on the efficacy of Ovsynch protocol in buffalo. Out of 686 buffalo heifers (HE) and 273 cows (BC), 8 heifers and 9 cows were used to monitor and evaluate ovarian follicular dynamics and serum progesterone profile during this protocol while the rest of animals were used to compare the conception rate following the application of this protocol, respectively. Total of 385 control buffalo heifers (CHE, n = 219) and cows (CBC, n = 166) were used as a reference for conception rate. The heifers and buffalo-cows were cyclic. All treated animals were injected with GnRH on Day 0, PGF2α on day 7, GnRH on day 9 and inseminated artificially 16 h later. Ovarian changes were monitored daily using ultrasound and serum progesterone (P4) in the investigated animals. All heifers (8) and 5 cows had F > 8 mm (LF) at the first GnRH injection. The first GnRH injection resulted in ovulation in 7/8 HE (87.5%) and 5/9 BC (55.5%). Following second GnRH, ovulation occurred in 100% of HE and 88.8% of BC. Ovulation started earlier in BC (10.41 ± 7.6 h) following second GnRH and extended for longer (22.6 ± 5.4 h) in HE. The average P4 concentrations of the HE were slightly greater than those of the BC on day 7 (P = 0.04). Conception rate in HE was 62.54 % (429/686) and was 59.82 % (131/219) in CHE, while it was only 22.71 % (62/273) in BC and 59.64 % (99/166) in CBC. It is suggested that the unsatisfactorily low conception rate in buffalo-cows -compared to heifersmay be attributed to the early ovulation and sub-functional CL.

The effect of parity on the efficacy ofan ovulation synchronization (O vsynch) protocol in buffalo (Bubalus bubalis)

Changes in blood flow in ovine follicles and serum concentration of estradiol 17 beta (E2) and nitric oxide (NO) around the time of ovulation in Ossimi ewes.

Contents
To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor-9 (GDF-9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF-9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen–thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF-9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF-9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF-9 was detected in mural granulosa cells and theca cells of pre-antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF-9. In corpora lutea, GDF-9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF-9 and cultured control that indicated the GDF-9 treatment has no effect on the primordial to primary follicle transition. GDF-9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre-antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF-9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF-9. In conclusion, treatment with GDF-9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).

T. M. El-Sherry

Papers

Characterization of rheotaxis of bull sperm using microfluidics

Development of computer-assisted sperm analysis plugin for analyzing sperm motion in microfluidic environments using Image-J.

The effect of parity on the efficacy ofan ovulation synchronization (O vsynch) protocol in buffalo (Bubalus bubalis)

Changes in blood flow in ovine follicles and serum concentration of estradiol 17 beta (E2) and nitric oxide (NO) around the time of ovulation in Ossimi ewes.

Effect of growth differentiation factor-9 (GDF-9) on the progression of buffalo follicles in vitrified-warmed ovarian tissues.