Validation of fetal microchimerism after pregnancy in the ovine using qPCR

Abstract

Fetal microchimerism has been detected in maternal tissues of humans and rodents during and after pregnancy. Studies focusing on fetal DNA transfer to maternal tissues in domestic animals are limited, especially in sheep. Fetal ram DNA was observed in the maternal circulation during pregnancy, but it is not known if this chimerism persists in soft tissues after parturition. The objectives of this exploratory study were to: 1) determine if male fetal DNA is detectable in soft tissues of mature ewes after parturition and if so, determine if detection repeatability differed with lifetime offspring sex ratio and 2) determine if male fetal DNA was present in soft tissues of yearling (primiparous) ewes shortly after parturition. Eight mature (open, non-lactating) and 8 yearling (primiparous, periparturient) Rambouillet ewes were used. Mature ewes (5- to 7-yr old) had given birth to primarily 82% males (n = 4) or 71% female (n = 4) over a lifetime. Yearling ewes had birthed either a singleton male (n = 4) or female (n = 4) lambs. DNA was extracted from 10 and 11 different soft tissues from the mature and yearling ewes, respectively. Real-time PCR (qPCR) was used to identify the presence of the SRY gene in each tissue sample. Male DNA was detected in the brain and liver from one mature open ewe that had given birth to two males and six females during her lifetime. In younger ewes that gave birth to a ram lamb, male DNA was observed in the thyroid of one ewe and the pancreas and brain of a second ewe. Male DNA was detected in the ovary of one ewe that had given birth to a female lamb. Based on these data, we suggest fetal microchimerism in soft maternal tissues is possible in sheep and may remain after pregnancy has ended. The detection repeatability of male fetal DNA was not associated with sex ratio of lifetime offspring. Male DNA was observed in maternal soft tissues collected shortly after parturition. The greater detection of fetal male DNA found in younger ewes shortly after parturition may be due to not having enough time for fetal DNA clearance to occur. Future studies are warranted to further study XY chimerism in maternal tissues of the ewe and its potential role in ovine physiology.

INTRODUCTION

The ability of fetal cells to cross over the placental interface into the maternal circulation during pregnancy has been studied extensively in humans and rodents (reviewed by Boddy et al., 2015). Most fetal cells are thought to be cleared by the maternal immune system after birth (Kolialexi et al., 2009), but a small number may remain in the maternal circulation or migrate to various tissues and result in microchimerism (Bianchi et al., 1996; Bayes-Genis et al., 2005). Even though fetal DNA was observed in tissues of healthy women (Bayes-Genis et al., 2005; Nelson et al., 2012), it was detected in higher frequencies in women with a variety of pathological conditions, especially autoimmune diseases (Nelson et al., 2012).

Fetal cell transfer into the maternal blood circulation was assumed to be more difficult in species with less invasive epitheliochorial placentas (Leiser et al., 1997; Chucri et al., 2010; Carter and Enders, 2013). Transfer of fetal cells from a male to a female co-sibling during pregnancy has been observed in cattle (Niku et al., 2007) and XY chimerism was identified in tissues and organs of female sheep (Keszka and Jaszczak, 1996; Keszka et al., 2001; Brace et al., 2008), goats (BonDurant et al., 1980; Szatkowska and Switonski, 1996), and pigs (McConico et al., 2011) born with male co-siblings.

In sheep, most studies associated with fetal microchimerism focused on transfer of fetal DNA between a male and female co-twin rather than movement to maternal tissues. Development of XY chimerism from the transfer of fetal DNA from a male co-twin to his female uterine counterpart occurred at a rate around 4% (Brace et al., 2008). The presence of XY chimerism in ewes may be used to diagnose freemartinism (Szatkowska, 1996; Szatkowska and Switonski, 1996), which impacts reproductive performance in ewes. Is transfer of fetal DNA from male offspring to maternal tissues possible in sheep, and if so, could this potentially affect the mother? Literature examining the transfer of fetal DNA to maternal tissues is very limited in domestic animals, especially in sheep. Cell free fetal DNA was detected in the maternal blood of ewes during pregnancy (Kadivar et al., 2013; Saberivad and Ahsan, 2016), but there are no published reports in ewes that examine fetal microchimerism in soft maternal organs or tissues after pregnancy has ended.

Using real-time quantitative PCR (qPCR) to detect the presence of the SRY gene in ewes, this exploration study addressed the following basic questions concerning the transfer of fetal DNA to maternal ewes: 1) Is male fetal DNA detectable in soft tissues of older ewes long after parturition? If yes, does detection repeatability differ with lifetime offspring sex ratio? 2) Is male fetal DNA present in soft tissues of younger ewes shortly after parturition?

MATERIALS AND METHODS

All methods and procedures described herein that involved animals were reviewed and approved by a USDA, ARS IACUC (#18-02). Animal husbandry, management, and handling procedures in the research environment were in accordance with the Ag Guide (Federation of Animal Science Societies, 2010).

Animals and Tissue Collection

Eight mature (5- to 7-yr-old) and 8 yearling (1-yr-old) Rambouillet ewes were used. Ewes originated from a wool-type flock managed in an extensive range-based system as previously described (Leeds et al., 2012; Notter et al., 2012). Mature ewes were neither pregnant nor lactating and had given birth within 8 to 21 mo. Four ewes had birthed primarily male (82%) offspring and four ewes had birthed primarily female (71%) offspring over their lifetime. Yearling ewes (primiparous) had given birth within 2 wk to either a singleton male (n = 4) or female (n = 4) lambs. Six yearlings were from primiparous yearling births and two were from multiparous births. All sheep were euthanized (lethal injection; AVMA, 2013) and a total of 20 mg of tissue from adrenal gland, brain, heart, kidney, liver, lung, mesenteric lymph node, ovary, pancreas, spleen, and thyroid were collected from animals at slaughter and frozen at −20 °C for further analysis.

PCR Controls

To provide quality controls, singleton male and female lambs from yearling ewes were used. Tissues from female lambs (n = 4) in addition to PCR grade water (NTC; n = 96) were used as negative controls and tissues from male lambs (n = 4) were used as positive controls. Control lambs were euthanized (lethal injection; AVMA, 2013) at the same time as yearling ewes and the same tissues were collected and processed.

DNA Extraction and Real-time qPCR

Each tissue sample was mixed with 200 µL of ATL buffer and Proteinase K solution and incubated overnight at 55 °C overnight. Tissues were lysed using a Quiagen TissueLyser II (Germantown, MD) and underwent QIAcube HT DNA extraction procedure. Quantity of DNA was determined using a NanoDrop spectrophotometer (ThermoScientific, Waltham, MA). Upon determining amount of DNA extracted, DNA samples were diluted to a uniform concentration of 25 ng/µL per sample.

Primers for the ovine SRY gene (forward: 5′-GACAATCATAGCGCAAACGA-3′ and reverse: 5′-CAGCTGCTTGCTGATCTCTG-3′) were synthesized by Integrated DNA Technologies (Coralville, IA) according to sequences published by Dervishi et al. (2008) and Saberivad and Ahsan (2016). Primers were optimized using a BioRad CFC Connect real time PCR thermalcycler (Hercules, CA) with the following conditions: 1 cycle at 95 °C for 2 min, 95°C for 15 s, with annealing temperature of 55 °C for 35 s. Melt curve analysis was performed after amplification and melting temperatures for the specific SRY amplicon were predicted by uMelt (University of Utah, Salt Lake City, UT). qPCR was conducted in 50 µL reaction volumes containing 25 µL 2× Go-Taq qPCR Master Mix (Promega, Madison, WI), 1 µL (0.2 µM) of each primer, 18 µL of DNA/RNAase free distilled water, and 5 µL of template DNA. Amplification for each DNA sample was performed in triplicate and all samples underwent electrophoresis in 2% agarose gel and stained with GelGreen (Biotium, Fremont, CA). To protect against contamination and false positives with the PCR product, samples were set up in a HEPA filtered laminar flow PCR workstation (Mystaire, Creedmoore, NC) and handling and processing of all samples were done by female technicians only to prevent potential contamination.

RESULTS

Optimization of qPCR

The amplification product for the SRY primer pair produced a single peak with melt curve analysis and had a melting temperature of 84.5 °C as predicted by uMelt (Figure 1). Gel electrophoresis resulted in a single band of 171 bp for male controls. Amplification efficiency was between 90% and 100% and sensitivity of the primer pairs was able to detect the SRY gene to 1.25 pg/µL, down to a serial dilution of 1/10⁴ (Figure 2). No false positive SRY PCR products were seen at any time in any negative control animals or water controls.

Figure 1.

Melt curve analysis of SRY amplicon during thermal cycling. The graph represents the plot of change in fluorescence with temperature (−d[RFU]/dT) plotted against the temperature in Celsius that produced the greatest change in fluorescence. The single peak represents the SRY amplicon produced by this primer pair, producing a single peak, whereas negative samples did not.

Figure 2.

Primer pair sensitivity in serial dilutions of ram DNA, 1/10 = 10-fold serial dilutions of 10; W = NTC PCR negative control, F = female negative control, L = 100 bp DNA ladder.

Lifetime Percentage of Male vs. Female Offspring from Mature Ewes

In mature open ewes that had a greater lifetime percentage of male births, no amplification products for the SRY gene were detected in any tissue sample (Table 1). In mature open ewes that had a greater lifetime percentage of female births, no positive PCR amplification products were detected in adrenal, heart, kidney, lung, mesenteric lymph node, and spleen. However, a PCR product of 171 bp for the SRY gene was detected in a single replicate of brain tissue and liver in one ewe (ID #R8425) that had given birth to two males and six females during her lifetime (Figure 3A and B). The original DNA extraction from the brain and liver samples that produced a positive result underwent qPCR 6 additional times to determine if additional 171 bp amplicons could be produced, but the SRY gene was not detected in any of the additional replicates from either tissue (Table 1).

Table 1.

Detection repeatability of positive PCR amplification products in mature and yearling ewes

Ewe ID # and Groupa,b	Sex of offspring	Tissue	Positive PCR product per replicate (repeatability)
R8425 mature open	Majority female	Brain	1 of 9 (0.11)
R8425 mature open	Majority female	Liver	1 of 9 (0.11)
Z5057 yearling periparturient	Male	Thyroid	9 of 9 (1.00)
Z5057 yearling periparturient	Male	Pancreas	6 of 9 (0.67)
Z4637 yearling periparturient	Male	Brain	4 of 9 (0.44)
Z5138 yearling periparturient	Female	Ovary	9 of 9 (1.00)

^aMature open = 5- to 7-yr-old ewe that had not given birth 8 to 21 mo prior to tissue collection.

^bYearling periparturient = 1-yr-old ewe that had given birth 2 wk prior to tissue collection.

Figure 3.

(A and B) Gel electrophoresis of SRY amplification in tissues and organs of ewe ID # R8425. L = 100bp ladder; M = male positive control; W = NTC PCR negative control; F = ewe negative control; A = adrenal; B = brain; H = heart; K = kidney; Li = liver; Lu = lung; Ly = mesenteric lymph node; P = pancreas; S = spleen; T = thyroid.

Yearling Ewes Giving Birth to a Male

A positive PCR amplification product of 171 bp for the SRY gene was detected in all 3 replicates of thyroid in 1 yearling ewe (ID #Z5057; Figure 4) and when the original DNA extraction from the thyroid was repeated an additional six times, a single positive amplification band of 171 bp was produced six out of six times (Table 1). A single band PCR amplification product of 171 bp was also observed in younger ewes with one replicate of the brain (ID #Z4637) and two replicates of the pancreas (ID #Z5057; Figure 4). When the original DNA extractions for the brain and pancreas from these ewes were repeated an additional six times, a single band of 171 bp was observed in three of the replicates of brain tissue and in four additional replicates of the pancreas (Table 1). No positive PCR amplification products were detected in adrenal, heart, kidney, liver, lung, mesenteric lymph node, and spleen in any yearling ewes giving birth to a male.

Figure 4.

Gel electrophoresis of SRY amplification in tissues and organs of young ewes. L = 100 bp ladder; M = male positive control; W = NTC PCR negative control; F = ewe negative control; B = brain of Ewe ID # Z4637; P = pancreas and T = thyroid of Ewe ID # Z5057; and O = Ovary of Ewe ID # Z5138.

Yearling Ewes Giving Birth to Female

A positive PCR product was detected in the ovary (ID #Z5138) of a yearling ewe that gave birth to a female lamb (Figure 4). When the original DNA extractions from the ovary were repeated, a single band of 171 bp was observed six out of six times (Table 1).

DISCUSSION

Most literature associated with fetal microchimerism in domestic animals was focused on movement of cells between co-siblings rather than the transfer between fetus and mother. Using sensitive PCR techniques and/or metaphase smears, many studies in sheep and goats observed male fetal cells in tissues and/or organs of females that developed with male co-siblings. For example, XY chimerism was identified in the blood (Keszka et al., 2001; Brace et al., 2008), liver, diaphragm, and ovaries of female ewes born with one or more ram co-siblings (Brace et al., 2008). The rate of fetal cell exchange did not differ between litters with different sex ratios or litter sizes (Brace et al., 2008). Male DNA has also been observed in bone marrow (BonDurant et al., 1980) and blood (Szatkowska and Switonski, 1996) in mature reproductively-normal and freemartin female goats born with male co-siblings. The repeatability of XY chimerism observed in female littermates in sheep and goats occurred at a rate of approximately 4% to 5% (Szatkowska and Switonski, 1996; Brace et al., 2008). Steinkraus et al. (2012) failed to observe any fetal cell transfer between transgenic goat co-siblings, and suggested that if transfer was possible in this species, it would occur at a low repeatability.

In the current study, transfer of male DNA to maternal tissues was most likely from connections between the fetal chorion and maternal endothelium that resulted in fetal cell transfer via tissue migration (McConico et al., 2011) or by anastomoses that may form between fetuses with epitheliochorial placental types such as cattle (Padula, 2005), goats (BonDurant et al., 1980), and sheep (Smith et al., 2000). The frequency of fetal cell transfer to maternal organs and/or tissues in domestic animals seems to vary among species. Fetal cell transfer was not observed in maternal tissues or organs in the pig (Garrell et al., 2014) and goat (Steinkraus et al., 2012), but seen in cattle (Turin et al., 2007) during pregnancy and after parturition. Studies of fetal microchimerism in sheep were focused on evaluating maternal blood collected during pregnancy. For example, fetal nucleic acids were observed in the circulation of ewes during pregnancy (Mandel and Matais, 1948) and concentrations of free-floating fetal cell DNA during early pregnancy were used as an alternative tool for offspring sex determination (Kadivar et al., 2013; Saberivad and Ahsan, 2016). No previous studies in sheep have examined the detection repeatability of fetal DNA transfer to maternal tissues post-partum.

Using a variety of soft tissues collected from open mature ewes and periparturient, primiparous yearling ewes, we optimized a qPCR reaction that was able to detect the presence of male fetal DNA. Male DNA was observed in the brain and liver at low repeatability in a mature ewe (5- to 7-yr-old) that gave birth to two males and six females over her lifetime. Considering the fact that 4 older ewes had a lifetime birth rate of >80% males, the results suggested that fetal cell transfer to maternal soft tissue is first, possible, and second, the number of male offspring a ewe gives birth to over her lifetime did not influence the repeatability of fetal cell transfer. This seems to agree with Brace et al. (2008), who reported that the sex ratio of co-siblings did not have an effect on the rate of XY chimerism of rare freemartin ewes.

Fetal male DNA was also observed in periparturient, primiparous ewes that had given birth to a ram lamb, with high detection repeatability in the brain, pancreas, and thyroid (Table 1). The higher detection repeatability may have been due to the recency of parturition (2 wk). Fetal cells were observed in the circulation of pregnant ewes (Kadivar et al., 2013; Saberivad and Ahsan, 2016), but the timing of fetal cell transfer to soft maternal tissues and subsequent residence time is unknown.

In humans, male fetal DNA was not only observed in the systemic circulation, but found in both parenchymal and hematopoietic tissues in women that carried male offspring and had died during pregnancy or shortly after partition (Rijnink et al., 2015). Distribution of fetal DNA reflected patterns of blood flow, with greatest quantities observed in the lungs and lowest in the brain (Rijnink et al., 2015). Concentration of human fetal DNA in the blood increased as pregnancy advanced (Lo et al., 2000) and cleared rapidly after parturition (Kolialexi et al., 2009). A similar pattern is thought to occur in the ewe, with greater amounts of fetal DNA present in the last 2.5 mo of gestation than the first 2.5 mo (Kadivar et al., 2013), but, as suggested earlier, the timing of fetal cell transfer into maternal organs or fetal DNA clearance from maternal tissues after parturition is unknown. It is possible that the higher repeatability of fetal DNA seen in soft tissues of periparturient, primiparous yearling ewes is simply a function of time since pregnancy. In other words, as time since parturition increases, clearance of fetal DNA is more likely. The lower repeatability of fetal microchimerism detected in mature ewes suggested that the presence of fetal cells that may have transferred to soft tissues during pregnancy or parturition decreased as the post-partum period increased.

Until this study, it was not known if male fetal cells were able to cross the blood brain barrier in domestic ruminants to result in fetal microchimerism. Fetal DNA has been found to cross the blood brain barrier in mice and humans. Male fetal DNA was observed in brain tissue of mice after pregnancy and associated with certain pathological conditions (Zeng et al., 2010). Low quantities of male fetal DNA were observed in the brain of women that had died during pregnancy or shortly after parturition that carried or gave birth to male offspring (Rijnink et al., 2015). Similar qPCR techniques that were used in this study quantified male-derived DNA post-partum in brain tissue of women (Chan et al., 2012) and a higher prevalence was seen in women with neurological disease (Chan and Nelson, 2013).

The low detection repeatability of male DNA observed after pregnancy in mature ewes was similar to what was reported in cattle (Turin et al., 2007). Using similar qPCR techniques, transgenic fetal DNA was detected in only one replicate of maternal blood samples in one recipient cow that received transgenic embryos 2 yr prior. Our exploratory study focused on detection of male fetal DNA using specific SRY primers rather than quantification of fetal cells. Now that we know male fetal DNA is present in tissues of older and younger ewes after parturition, future studies can focus on determining fetal DNA concentrations related to cell quantity and/or gene copies in soft tissues and how they relate to physiological mechanisms.

The origin of fetal DNA located in the brain and liver of the mature ewe was not known. Twenty-five percent of her offspring (2) were male lambs, with the last ram lamb born 2 yr prior to tissue collection, so it is likely the male fetal cells were from her male offspring. However, the mature ewe was also born co-sibling to two rams as a member of a triplicate birth. XY chimerism has been observed in ewes born with a male co-sibling (Szatkowska and Switonski, 1996; Keszka et al., 2001; Brace, 2008) at a rate of 4% (Brace et al., 2008), but whether or not the XY chimerism affected estrus and/or reproducing in these ewes was unknown. A very low percentage of ewes are born as freemartins, ranging from 1% to 6.8% (Marcum, 1974; Keszka et al., 2001; Padula, 2005) and ewes born with a male co-twin had a reproductive advantage, with greater lifetime weaning weights (Brown et al., 2016). The mature ewe in the present study was able to successfully reproduce 6 out of 7 yr (at ages 2 to 7) and produced live offspring each year. Based on the data and history of the ewes, we are not able to definitively determine if the male DNA is of fetal origin, but results verify that our technique used to detect male DNA in maternal soft tissues was valid. The 2 yearling ewes that gave birth to a ram and had positive PCR products (ID #Z4637 and #Z5057) were born as singletons from primiparous yearlings. Thus, the male DNA detected in soft tissues of these younger ewes is suggested to be of fetal origin.

One yearling ewe gave birth to a female lamb and had a detectable amount of male DNA in her ovary, with consistent repeatable positive PCR amplicons. This primiparous ewe was born with a female co-twin and her dam had given birth to male offspring in 2015 and 2013, thus there is a chance the male DNA could have crossed from the dam to her circulation to colonize into her ovary. In humans, male fetal cells from previous pregnancies were able to survive long periods of time in the maternal circulation and transfer to soft tissues of subsequent female siblings (Guettier et al., 2005).

Conclusion

In conclusion, this exploratory study is the first to demonstrate that XY chimerism after pregnancy in soft tissues of ewes is possible. Male fetal DNA was detected at a high repeatability in some soft tissues shortly after parturition, which was similar to what has been reported for humans (Bianchi et al., 1996; Bayes-Genis et al., 2005; Rijniki et al., 2015). Fetal microchimerism after pregnancy seems to be possible in sheep. Occurrence was rare, nevertheless, and was not associated with the birthed sex ratio over a lifetime. Clearance of fetal DNA from maternal soft tissues may also change over time during the post-partum period. If fetal microchimerism affects lifetime performance in ewes, producers may be able to affect outcomes by modifying ewe retention. However, further research is warranted to determine patterns and quantities of fetal cells that transfer to maternal tissues and colonize after pregnancy and their potential role in ovine physiology.