The assisted reproductive technology of in vitro embryo production

Reproductive efficiency is the cornerstone of all animal-based agricultural enterprises and is crucial for profitable, environmentally sustainable food systems. In livestock production systems, particularly cattle production, reproductive efficiency is the main driver of farm profitability. Pregnancy loss, rather than fertilization failure, is one of the major causes of reproductive failure in cattle [1, 2] and leads to extended calving intervals which, especially in a seasonal system, can have a major impact on profitability due to costs associated with increased calving intervals, increased culling, increased labor costs, and increased interventions of one form or another [3].

In cattle, most pregnancy failure occurs quite early after fertilization; ∼75% of conceptus loss occurs in the first 2–3 weeks of gestation, before maternal recognition of pregnancy (around day 16–17) and the start of placentation (around day 20) [2, 4-7]. Indeed, in some situations (e.g., metabolic stress associated with high milk production), as many as 50% of embryos may be lost in the first week after fertilization [4, 6]. Even when all of the biological and technical causes for pregnancy failure in the first week are avoided by transferring an embryo directly into the uterus (typically done on day 7 of the cycle), pregnancy success is not consistently improved compared to artificial insemination (AI) [8]. Thus, improving our understanding of the underlying physiological and molecular regulation of early embryo development leading to a successful pregnancy will significantly contribute to social and economic sustainability in agri-food production, a crucial objective in the face of an ever-increasing global population [9] and growing concerns about the impact of inefficient agricultural practices on the environment [10].

The development of AI in the 1950s has driven genetic improvement in dairy cattle and is now the main method of impregnating dairy females with semen from elite bulls [11]. Since then, considerable progress has been made in the development and application of a wide range of assisted reproductive technologies (ARTs) at farm level [12], including multiple ovulation embryo transfer (MOET, or ‘superovulation’), involving the generation of multiple embryos within the female (in vivo) [13], ovum pick-up/in vitro fertilization (in vitro embryo production), involving the generation of embryos in the laboratory [14, 15], and the use of sex-sorted semen to predetermine the offspring sex [16-18]. All these technologies facilitate accelerated genetic improvement and increase the economic value of the offspring generated [19].

In vitro embryo production (IVP) is now an established technology in the toolbox of ARTs available to farmers and breeding companies for genetic improvement in dairy cow herds. The number of IVP bovine embryos transferred annually has increased year-on-year in the last decade and now surpasses the number derived by traditional superovulation, accounting for approximately 80% of all bovine embryos produced and transferred globally [20]. According to the latest data available from the International Embryo Technology Society (www.iets.org), over 1.6 million IVP bovine embryos were transferred commercially worldwide in 2022 compared to ∼394,000 in vivo-derived embryos, and this number is predicted to continue to increase [20]. IVP offers significant advantages over traditional MOET including increased flexibility in sire usage allowing multiple pregnancies from elite dam–sire combinations to be generated, the ability to produce more embryos per unit time per genetically elite female, the ability to use oocytes from prepubertal females (as young as 2 months of age) to reduce the generation interval (‘Juvenile In Vitro Fertilization ET’, JIVET) [21], and the more efficient use of rare or high-cost semen straws (e.g., sex-sorted semen) as many oocytes can be inseminated with one semen dose [19]. The increased use of sex-sorted semen to breed replacements and beef semen to improve calf quality in dairy herds [22] will ultimately lead to a reduction in the pool of male dairy calves available as future potential AI sires [23]. Under such conditions, the targeted use of IVP with elite breeding stock will facilitate the production of future generations of elite bulls. Despite these major benefits of IVP technology, significant challenges relating to pregnancy loss after ET, particularly after cryopreservation (freeze/thawing) of IVP embryos, and issues relating to peri and postnatal health and development of IVP offspring remain to be resolved and hamper the more widespread application of the technology.

Approximately 80%–90% of immature bovine oocytes submitted to IVP undergo nuclear maturation in vitro and reach metaphase II, about 80% undergo fertilization, 30%–40% develop to blastocyst stage, and around 50% of transferred embryos establish a pregnancy [8, 14]. Improvements in culture media and IVP processes have improved initial pregnancy rates to the extent that they are now comparable with AI when transferred fresh; however, subsequent pregnancy loss, particularly from frozen-thawed IVP embryos, remains an obstacle to their more widespread use [5, 8, 24, 25]. We recently quantified the timing and incidence of pregnancy loss following AI or ET with fresh or frozen in vitro produced embryos [25]. Pregnancy was diagnosed in cows that had not been detected returning to estrus by quantification of the mRNA abundance of interferon-stimulated gene-15 (ISG15) in maternal peripheral blood on day 18, concentration of pregnancy specific protein B (PSPB) in maternal serum on day 25, and transrectal ultrasonography on days 32, 62, and 125, respectively, after synchronized ovulation, and finally by recording a parturition event at full-term. Results illustrate that most embryonic loss occurs early after fertilization; the largest proportion of pregnancy loss occurred before day 18. Pregnancy loss from day 32 to day 62 was greater following transfer of an IVP embryo compared with AI, particularly with frozen embryos, while losses after day 62 were small (≤3.5%).

The underlying mechanisms responsible for such loss are not clear but are likely related to the consequences of suboptimal postfertilization culture conditions on blastocyst quality [26-28]. IVP embryos differ in terms of morphology, ultrastructure, cryotolerance, and transcriptome from those derived in vivo [14], leading to a compromised ability of the resulting conceptus to appropriately signal to the maternal endometrium during elongation and attachment [29-31]. Appropriate molecular interaction between conceptus and endometrium is an important feature in the period leading up to attachment [29, 32, 33], and dysregulation of conceptus–maternal communication is a probable cause of lower calving rates after the transfer of IVP embryos.

Emerging data indicate that the time to conceptus attachment is a direct determinant of subsequent pregnancy loss in lactating dairy cows [34-36]. In cattle, attachment typically first occurs around day 20–21 postfertilization [37]. During this period, trophoblast giant binucleate cells develop within the chorion to migrate and fuse with the uterine surface epithelium to form syncytial plaques. These binucleate cells produce pregnancy-associated glycoproteins, including pregnancy-specific protein B (PSPB), which migrates from the conceptus across the newly forming placenta into maternal circulation [38, 39]. Recent studies have highlighted that the timing of presumptive conceptus attachment (pCA), as assessed by increasing concentrations of PSPB in maternal circulation, is strongly associated with subsequent pregnancy loss in lactating dairy cows [34, 36, 40]. In cows that had conceptus attachment later than day 21 post-ovulation, the likelihood of pregnancy loss was four times greater compared with cows that had conceptus attachment on day 20 or 21 [36]. Our recent data on the timing of pCA and incidence of pregnancy loss in lactating dairy cows following AI or transfer of a frozen-thawed IVP embryo indicate that the timing of conceptus attachment, as measured by a sustained increase in PSPB, is later followed by the transfer of an IVP embryo compared with AI and is associated with increased risk of pregnancy loss between day 30 and day 60 [41]. Serum PSPB was measured on day 17 (baseline) and day 19 through day 28 after ovulation to characterize the increase in concentrations believed to occur in conjunction with pCA. The day of pCA was defined as the first day of an increase in PSPB of ≥12.5% from baseline followed by two more consecutive days of ≥12.5% increase from the previous day. The day of pCA was earlier following AI compared with IVP-ET. Calving/service event was greater (83.2% vs. 54.4%) and pregnancy loss during the interval from pCA to expected calving date was less (16.8% vs. 45.6%) for cows with early pCA (≤ d 20, 23/137) compared with cows that had late pCA (≥ d 21, 36/79). Furthermore, the incidence of pregnancy loss was greater for cows assigned to IVP-ET (33.8%) than AI (16.4%).

Continual improvements in culture media used for the in vitro production of bovine embryos coupled with a better understanding of the characteristics of a competent embryo will contribute to improved pregnancy rates and reduced pregnancy loss.

Pat Lonergan: Writing—review & editing; writing—original draft.

The authors declare no real or perceived conflicts of interest.