Intracytoplasmic sperm injection- produced equine embryos: transport, thawing, and transfer Rob Foss Equine Medical Services, Inc., Columbia, MO Abstract Intracytoplasmic sperm injection (ICSI)-produced equine embryos are transferred at the early blastocyst stage, similar to a day 6.5 in vivo-produced embryo and will benefit from a recipient mare that ovulated 3 - 5 days before transfer. Frozen-thawed and vitrified-thawed embryos may have an even narrower window of synchrony with recipients for best results. Day of blastocyst for- mation is a useful indicator of embryo quality and potential for establishing a successful pregnancy; early embryonic loss is higher for blastocysts that formed on days 9 and 10 after ICSI than those formed earlier. Keywords: Intracytoplasmic sperm injection, equine embryo, embryo transfer Introduction Production of equine embryos via intracytoplasmic sperm in- jection (ICSI) and their subsequent transfer is becoming more important for commercial horse production. Many of these embryos are potentially valuable and contain genetics prized by their owners, so it is in the best interest of veterinarians and clients to handle and transfer these embryos as optimally as possible. Intracytoplasmic sperm injection-produced em- bryos (hereafter referred to as ICSI embryos) differ from in vivo-produced embryos. Although they can be handled sim- ilarly, some adjustments are necessary. Careful selection of appropriate recipient mares, embryo handling, and timing of transfer are necessary to optimize results. Comparison of in vivo and intracytoplasmic sperm injection-produced embryos Equine ICSI embryos differ from their in vivo counterparts morphologically and metabolically.1 In vitro culture condi- tions can be successful for viable embryo production; howev- er, conditions are not similar to those that exist in utero (uter- us or oviduct), leading to many differences. One of the more prominent morphological differences of ICSI embryos is the lack of development of an embryonic capsule. The capsule, a mucin-like glycoprotein, is normally produced from secre- tions from the trophoblast cells, and forms between them and the zona pellucida. A very strong, elastic structure, it presum- ably provides protection for the developing blastocyst after shedding zona pellucida. A capsule does not form on ICSI embryos in vitro; however, it develops after embryos are trans- ferred into a recipient uterus. Due to the injection-induced breach in the zona, there is a short interval after transfer when ICSI embryos may be directly exposed to the uterine immune system and therefore inflammation. The discrete inner cell mass that develops in the early blasto- cyst in vivo is not observed in ICSI embryos, another result of in vitro culture. The delay in the aggregation of inner cell mass cells may contribute to the higher rate of monozygotic twinning after transfer of equine ICSI embryos. Zona pellucida appears to be somewhat hardened by in vi- tro culture. This has a role when the early blastocyst starts ex- panding and is limited in its expansion by the zona. During in vivo development, the zona thins as the blastocyst expands and then, after the capsule forms, thins enough until it flakes away. During in vitro, the zona does thin; however frequently the trophoblast layer will herniate through the ICSI-generated hole in the zona. This, when the embryo is transferred, would further expose the otherwise unprotected blastocyst to the uterine environment. Herniation of trophoblast and blasto- coele might also predispose to monozygotic twinning, form- ing a separate blastocyst outside the zona pellucida. An ICSI embryo, growing in culture instead in an oviduct, is subjected to physiologic stress throughout the culture period. Without the conditions provided by oviductal epithelium, the various cytokines and other ‘embryo-friendly’ compounds, embryos do grow; however, they are probably compromised by the stress of adapting to in vitro culture conditions. There are differences in gene activation of the ICSI embryo com- pared to in vivo embryos.2 Problems with chromosomal segregation are more common in vitro leading to embryos with ploidy issues, i.e., too many or too few chromosomes in daughter cells.3 All of these factors contribute to the fact that at our current state of knowledge, ICSI embryos, on average, are less likely to establish a pregnancy and then maintain a viable pregnancy to term than their in vivo counterparts. Au- thor’s practice advises clients to expect a 10% lower pregnancy rate with ICSI embryos versus in vivo embryos, and approxi- mately twice the rate of early embryonic loss. Transfer of ICSI embryos Transfer of ICSI embryos is usually done at the early blastocyst stage as it is starting to expand but before much expansion occurs. This permits transfer of a uterine stage embryo that has not had trophoblast cells herniate through the breach in the zona. Currently, developmental status of the ICSI embryo is roughly equivalent to that of a day 6.5 in vivo embryo, al- though the embryo may be anywhere from 6 to 10 days after fertilization by ICSI. Synchrony Selection of a recipient mare to receive an ICSI embryo should consider the developmental stage of the embryo. Preferred synchrony for in vivo days 7 and 8 embryos is usually con- sidered ideal at -1 or -2 (a recipient that is on day 5 or 6 after ovulation), but with a window of +1 to -3 (recipient days 8 - 4). This suggests that the preferred recipient for an ICSI early blastocyst would be a recipient that was days 4 or 5 postovu- lation. Reports on the success of ICSI embryo transfer have suggested that the window of synchrony for transfer is narrow- er for ICSI embryos than their in vivo counterparts, although these reports utilized frozen-thawed embryos.4,5 Author’s clin- ical practice has not identified a narrower window of accept- able synchrony for embryos that have not been frozen. Data from 926 transfers (Table 1) suggested a trend toward higher pregnancy rates with day 3, 4, and 5 recipient mares and no statistical difference with days 2 through 7 recipients. Table 1. Pregnancy by day after recipient ovulation Recipient day Pregnant/transferred Pregnant (%)* Noncyclic 21/25 84.0 a 7 45/58 77.6 a 6 146/181 80.6 a 5 225/274 82.1 a 4 229/278 82.3 a 3 86/105 81.9 a 2 3/5 60.0 a Total 755/926 81.5 *Within a column, percentages without a common superscript differed (p < 0.05) Day of development Day of development, representing the number of days follow- ing ICSI that blastocyst formation occurs, has a distinct effect on results of embryo transfer. As expected, on average, the fast- er an embryo reaches the early blastocyst stage the healthier it is and the more likely to produce a viable pregnancy. Data (Ta- ble 2) from the same group of 926 transfers were compared by day of blastocyst development and pregnancy rate. A fairly consistent trend of decreasing pregnancy rate with increasing days of development was evident; however, the difference was not significant until days 9 and 10. Table 2. Pregnancy by day of blastocyst development Day Pregnant/transferred Pregnant (%)* 6 36/39 92.3 a 7 288/348 82.7 a 8 232/278 83.5 a 9 147/185 79.5 a,b 10 52/76 68.4 b Total 755/926 81.5 a,bWithin a column, percentages without a common superscript differed (p < 0.05) Early embryonic loss Early embryonic loss is more common with ICSI embryos than with in vivo embryos; author’s practice informs the cli- ents to expect ~ double the rate of embryonic loss. Most of these losses occur before day 30 of pregnancy and are usual- ly preceded by a period of slower than expected embryonic growth or they appear as an empty trophoblastic vesicle. There is a smaller group that appear normal at the heartbeat stage that are lost by 45 days, and an even smaller group that is lost by 60 days. Early embryonic losses for the group of 926 trans- fers (discussed previously) by recipient mare day postovula- tion are summarized (Table 3). The overall total loss rate was 17.7% with no significant differences related to synchrony. Table 4 has the same group classified by day of blastocyst de- velopment. A consistent trend in increased loss with increased day of development was noticed, becoming significant by days 9 and 10 post-ICSI. Table 3. Early embryonic loss by day of recipient cycle Recipient day Pregnant/transferred %* Losses %* Noncyclic 21/25 84.0a 2/21 9.5a 7 45/58 77.6a 10/45 22.2a 6 146/181 80.6a 24/146 16.4a 5 225/274 82.1a 46/225 20.4a 4 229/278 82.3a 35/229 15.2a 3 85/105 81.9a 14/86 16.2a 2 3/5 60.0a 3/3 100a Total 755/926 81.5 134/755 17.7 *Within a column, percentages without a common superscript differed (p < 0.05) Table 4. Early embryonic loss by day of blastocyst development Day Pregnant/transferred % Loss % 6 36/39 92.3a 4/36 11.1a 7 288/348 82.7a 34/288 11.8a 8 232/278 83.5a 35/232 15.1a 9 147/185 79.5a,b 41/147 27.8b 10 52/76 68.4b 20/52 38.5b,c Total 755/926 81.5 134/755 17.7 a-cWithin a column, percentages without a common superscript differed (p < 0.05) Grading ICSI embryos Traditionally embryos have been graded by evaluating mor- phological characteristics at a single point in time with the goal of providing useful information concerning the estimat- ed ability of a given embryo transfer to result in an ongoing pregnancy. Similar morphologic evaluation of ICSI embryos generally does not give the same ability to estimate the preg- nancy potential of a given embryo. ICSI embryos are trans- ferred at an early blastocyst stage that will appear remarkably similar for vigorous healthy embryos and those that are not so vigorous and healthy. Day of blastocyst formation/transfer has a higher correlation to pregnancy than morphology. Tables 4 and 5 suggest that early embryonic loss and pregnancy rate decreased with increasing day of blastocyst formation. Since owners and veterinarians alike want to know the grade of an individual embryo, author’s practice grades embryos based on day of blastocyst formation, adjusted for morphologic ab- normalities. Typically, days 6 and 7 blastocysts are graded as Grade 1, day 8 blastocysts are graded as Grade 1.5, Grade 2 for day 9, and Grade 2.5 for day 10. History noted during the culture period may also be used to adjust grades. Blastocyst formation normally occurs 2 days after morula compaction and if this period is longer, the embryo will be graded lower. Table 5. Pregnancy for vitrified/thawed embryos by day of re- cipient cycle Recipient day Pregnant/transferred Pregnant (%) Noncyclic 11/15 73.3 a 7 4/6 66.6 a 6 5/12 41.7 a 5 62/81 76.5 a 4 115/157 73.2 a 3 23/28 82.1 a Total 220/299 73.5 *Within a column, percentages without a common superscript differed (p < 0.05) Monozygotic twins Occurrence of monozygotic twins is more likely following transfer of ICSI embryos than in vivo embryos with an inci- dence of ~ 1.5%.6 These twin pregnancies occur following the transfer of a single embryo and are typically monochorionic, so that only a single vesicle is seen on initial pregnancy ex- aminations. A diagnosis for twin pregnancy is not performed until more than 1 embryo-proper is visible in the vesicle. Twin reduction of monozygotic twins by cranio-cervical dislocation is possible but generally not successful,7 so it is often prudent to consider this a pregnancy loss and abort the pregnancy. It is interesting to note that monozygotic twinning in human IVF embryos occurs ~ 1.7 - 2.5%,8 the lower rate from transfers on day 2 or 3 with the higher rate from transfers on day 5 or 6. This is an indication that it is the culture environment that leads over time to an increased incidence of twinning. This may be from ineffective migration of inner cell mass cells leading to the formation of two inner cell masses. Twin pro- duction via herniation of trophoblast with some inner cell mass cells through the Piezo hole in the zona is a less likely explanation as 2 embryonic vesicles should be formed in this manner leading to a dichorionic twin pregnancy. Shipping of ICSI embryos Since ICSI laboratories are often geographically distant from recipient mares, shipping becomes necessary. The ICSI em- bryo, being produced in only an imitation of its natural en- vironment, may be considered stressed, so additional stress should be avoided. Stresses to consider are temperature, du- ration of transport, and selection of culture/holding medium providing pH maintenance and embryo nutrition. Tempera- ture or osmotic stress are also a possibility, if the embryo is moved to a different medium (after shipping) before transfer. ICSI embryos are generally transported in passive heating/ cooling devices, such as the EquOcyte (Hamilton Biovet, Ip- swich, MA), or portable incubators capable of active heating and cooling (e.g., Micro Q [Micro Q Technologies, Scottsdale, AZ]). Equine ICSI embryos can tolerate several hours at room temperature, but pregnancy rates decreased as the interval at room temperature increased.7 Addition of nutritional support in the form of 10 mM glucose can increased the survival time, up to 50% pregnancy rate at 48 hours.7 Currently, author’s practice utilizes a modified culture medium for shipping in an incubator (Micro Q) at 38°C. The shipping medium can be used for transfer of the embryo so that temperature and osmotic changes are avoided, and an attempt is made to min- imize transport time as much as possible. Thawing and transfer of vitrified embryos Vitrified ICSI embryos can be thawed and transferred in most situations and practices that can transfer fresh embryos. Due to the number of embryos produced in the nonbreeding sea- son and surplus embryos produced during the breeding sea- son, many are vitrified. Vitrification gives an opportunity not only for storage but also for shipping and transfer into a spe- cific recipient or even back into the donor. Author’s practice has utilized thawing into a 0.3 M sucrose solution with timed stepwise reduction in sucrose concentration for several years, but have utilized a single step procedure for the last 2 years.9 This procedure consists of plunging the device containing the embryo (e.g., Cryolock [Bio Tech Inc., Alphareta, GA]) or open pulled straw, directly from liquid nitrogen into a petri dish containing the thawing solution warmed to 38°C. This thaw- ing solution is GMOPS Plus (Vitrolife, Goteborg, Sweden) in the author’s practice but commercial embryo holding medi- um can also be used. Embryo is held in the thawing solution on a warm stage for 5 minutes and then either prepared for transfer or placed in culture. If a specific recipient mare is to be used, directly transferring the embryo may be preferable, but if multiple recipients are available, culture for a short interval may be preferable as it allows evaluation of embryo re-expan- sion. Embryo re-expansion will take place rather quickly, 2 - 3 hours, in most instances but other embryos that may not have tolerated the freezing as well may take a day or even 2. This can drastically affect embryo-recipient synchrony, so culturing improves recipient selection in those instances. There is also a difference in timing between embryo re-expan- sion and resumption of growth. Embryonic vesicles detectable via ultrasonography on the first day of pregnancy are general- ly smaller or even a day later than expected from transfer of fresh ICSI embryos. This delay in resumption of growth may explain the narrower window of synchrony reported for trans- fer of frozen-thawed ICSI embryos.4,5 Results (Table 5) from transfer of vitrified-thawed embryos in the author’s practice broken down by recipient mare’s day of cycle. There is a trend toward higher pregnancy rates from days 3, 4, and 5, but it does not reach the level of significance. This may be since the majority of these embryos were cultured until re-expansion versus directly transferred into recipients. Conflict of interest None to declare. References 1. Tremolada J, Stout T, Lagutina I, et al: Effects of in vitro production on horse embryo cytoskeletal charactereistics and blastocyst capsule formation. Biol Reprod 2003;69:1895-1906. 2. Hisey E, Ross P, Meyers S: A review of OCT4 functions and applications to equine embryos. J Equine Vet Sci 2021;99:103394. 3. Ducheyne K, Rizzo M, Cuervo-Arango J, et al: In vitro production of horse embryos predisposes to micronucleus formation, wheras time to blastocyst formation affects likelihood of pregnancy. Reprod Fert Dev 2019;31:1830-1839. 4. Cuervo-Arango J, Claes A, Stout T: In vitro-produced horse embryos exhibit a very narrow window of acceptable recipient mare uterine synchrony compared with in vivo-derived embryos. Reprod Fertil Dev 2019;31:1904-1911. 5. Cuervo-Arango J, Claes A, Stout T: The recipient’s Day after ovulation and the number of corpora lutea influence the likelihood of pregnancy in mares following transfer of ICSI frozen embryos. Theriogenology 2019;135:181-188. 6. Djikstra A, Cuervo-Arango J, Stout T, et al: Monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos. Eq Vet J 2020;52:258-261. 7. Foss R: Unpublished data. 8. Kanter J, Boulet S, Kawwass J, et al: Trends and correlates of monozygotic twinning after single embryo transfer. Obstet Gynecol 2015;125:111-117. 9. Canesin H, Ortiz I, Filho A, et al: Effect of warming method on embryo quality in a simplified equine embryo vitrification system. Theriogenology 2020;151:151-158. ytoplasmic