Blood Sampling and Hormone Measurement for Determining the Stage in the Ovarian Cycle in Marmosets

The common marmoset (Callithrix jacchus) is a small New World monkey with many characteristics similar to those of humans, and the duration of its ovarian cycle is 26-30 days^1,2. Studies on the early development and the generation of genetically modified marmosets require the harvesting of oocytes and zygotes at specific stages in the ovarian cycle. Thus, accurate determination of the stage is crucial and can be estimated by measuring the blood levels of the hormones progesterone (P4) and estradiol (E2)^2,3. These hormones promote endometrial growth, which is necessary for implantation. P4 is produced from the corpus luteum, which forms in ovaries immediately following ovulation. E2 is secreted by the ovarian follicles in response to follicle-stimulating hormone (FSH) from the hypothalamus-pituitary complex in the brain. E2 levels increase as the follicle matures, peaking before ovulation³. High E2 levels cause the pulsed release of luteinizing hormone (LH) via the hypothalamus-pituitary complex in humans; this LH surge induces ovulation. However, in marmosets, the LH gene underwent degeneration during evolution, and ovulation is instead induced by the release of chorionic gonadotropin (CG), which has a similar structure to LH's, from the pituitary gland^4,5.

The ovarian cycle can be controlled by hormone injections. FSH injections, in humans, act on ovarian FSH receptors and are used to promote estrogen synthesis and follicle growth⁶. The injection of human CG (hCG) as a substitute for LH at the end of the follicular phase is used to stimulate ovulation in humans⁷. CG injections are also used to treat human infertility because CG stimulates the corpus luteum in early pregnancy, resulting in increased P4 production. Prostaglandin F2α (PGF2α) injections reset the ovarian cycle⁸. In domestic cattle, PGF2α injection is used to shorten the luteal phase and synchronize the estrus cycle for reproductive management.

Although marmosets and humans have similar biological mechanisms, making them ideal model animals, there is a lack of literature describing basic methods for many often-used techniques. Blood sampling is one of the most often used techniques^9,10,11,12. However, beginners sometimes have trouble finding the vein. Hence, this study conducted anatomical analyses of the femoral vein region. Based on anatomical observations, this protocol introduces the proximal region of the femoral triangle as an easy site for venipuncture.

All methods involving marmosets utilized high ethical and welfare standards and were approved by the Institutional Animal Care and Use Committee at the National Center for Child Health and Development. Animals used here were single-housed or paired-housed (one female and one male) with 12 h of light per day.

1. Blood sampling

1. Prepare a 1 mL syringe (the short type is easy to use) with a 25 G needle attached blade-side up. To avoid blood clogging, heparinize the syringe by drawing 200 µL of undiluted heparin sodium solution into the syringe. Coat the inside of the syringe evenly by pulling it up and down several times; then, expel the heparin solution from the syringe.
   NOTE: Since changing to a new syringe is often necessary, prepare a few additional heparinized syringes.
2. Prepare absorbent cotton and alcohol swabs. Switch on an adjustable lamp to illuminate the area where the marmoset will be placed for blood sampling.
3. Prepare the restraint device (420 x 85 x 85 mm, Figure 1A), which is commercially available (see Table of Materials for details). To place a marmoset into the device, open the retention part with the sponge belt, which secures marmosets. Insert the marmoset into the restraint device facing up.
   NOTE: It is not necessary to adapt marmosets to the restraint device. Marmosets are usually calm in the device.
4. Capture the marmoset; insert the marmoset into the cylindrical part; and secure it by pressing down the sponge belt. Place the leg from which blood is to be collected on top of the other (Figure 1A). Hold the legs using the non-dominant hand; place the middle and ring fingers inside each leg to fasten them and the other fingers outside each leg to fix them.
   NOTE: If a restraining device is not present, perform blood collection under anesthesia or with another person restraining the marmoset. When restrained, the marmoset's leg must not move during blood collection.
5. Check if a vein is visible near the base of the thigh. If not, palpate to find the pulsating artery and use it as a landmark to check that a vein runs inside it (Figure 1B-D).
   NOTE: Desk illumination and hair shaving are recommended to improve visibility. Visibility can also be improved by rubbing with alcohol swabs. Lymph nodes in the triangle are often located close to the vein and show a dark blue color as a vein. How to distinguish them is described in the Representative Results section.
6. Disinfect the puncture site using an alcohol swab. Insert the needle blade-side up at an angle of 15°-20°. To prevent the needle from slipping out of the blood vessel during blood collection, stabilize the hand holding the syringe, for example, by resting it on the other hand.
7. Gently pull back the plunger to apply negative pressure (Figure 1E). Push the needle tip forward. Once blood enters the syringe, maintain the position of the needle tip until the required volume (500-700 µL) has been collected.
   1. When blood does not enter the syringe, change the site of puncture. If the blood comes out when pulling out the needle, stop bleeding by applying pressure to the puncture site for 3 min. After stopping the bleeding, restart the venipuncture.
   2. If the blood being drawn into the syringe stops flowing during the drawing process, slowly push the needle tip forward and then pull it back to find the blood vessel. This may restore the flow of blood into the syringe. If not, pull out the needle and perform venipuncture using a new syringe.
      NOTE: Use the thigh on the other leg when it is difficult to collect blood from the same side.
8. Carefully pull out the needle while pressing lightly on the puncture site using the little finger. Then, using an absorbent cotton swab, immediately apply pressure to the puncture site for 3 min to stop the bleeding. Invert the syringe to mix blood and heparin.
   NOTE: Apply pressure for a longer time (5 min) while cooling when arterial blood is drawn, and carefully confirm the ceasure of bleeding to prevent the formation of hematoma, which can sometimes result in a fatal outcome.
9. After confirming that bleeding has ceased, remove the sponge belt, hold the animal's waist using one hand, and rotate the animal so that one can hold the animal's underarm using another hand from the backside.
10. Return the marmoset to its cage. To reduce stress and facilitate repeated blood sampling, provide the marmoset with its favorite food (e.g., biscuits, marshmallows, or sponge cake). Check the occurrence of hematoma occasionally.
    NOTE: When hematoma is found at the early stage, apply a pressure bandage to prevent the progression of hematoma. When it is found after a long time, surgical removal of hematoma with ligation of the femoral artery and blood transfusion may be needed^9,13.
11. Detach the needle from the syringe to prevent hemolysis. Then, slowly expel the collected blood along the inside wall of a 1.5 mL microtube.
    NOTE: The collected blood can be stored at 4 ^oC for up to 24 h before plasma separation for measuring P4/E2 levels.

2. Separation of plasma and determination of hormonal levels

1. Centrifuge the blood in a 1.5 mL tube at 1,100 × g for 5 min at 4 ^oC.
2. Transfer the separated plasma (supernatant) from the 1.5 mL tube to a new tube/cup, carefully avoiding the inclusion of blood cells (sediment).
3. Measure the P4 and E2 levels using an ELISA kit or an automatic analyzer. If the amount of plasma is not enough for the automatic analyzer, use a sample dilution solution.
   NOTE: For the measurement using an automatic analyzer, >175 µL of plasma is required for determining only a P4 level, and >250 µL of plasma is required for determining both P4 and E2.

3. Urine CG measurement to detect ovulation and pregnancy

NOTE: CG levels in marmosets can be measured using an immunochromatographic kit test for both ovulation and pregnancy. In the case of ovulation, a positive result can be obtained 0-2 days before ovulation. In the case of pregnancy, a positive result is detected from days 15-20 to approximately day 100 of pregnancy. The test requires a small amount (90 µL) of urine (one drop of urine is ~30 µL).

1. Tray method: Put the washed tray at the bottom of the cage to collect urine. Collect urine just after lighting up the room in the morning because urine is stored in the bladder. If two or more animals are in the same cage, separate the target marmoset (or other animals) into another cage in the morning.
   NOTE: Urine can be usually collected within approximately 30 min.
2. Squeezing method: Prepare the washed trays for urine collection. After locating the marmoset's bladder, carefully squeeze it from the front and both sides using the entire length of the fingers (Figure 1F). Collect urine just before lighting up the room in the morning.
   NOTE: If urine collection is unsuccessful due to the absence of urine in the bladder, wait a while and try again. Be careful not to use excessive force because this will injure the animal.
3. Immediately after collection, place the urine sample in the well of the immunochromatographic test kit. Read the result after 10 min according to the manufacturer's instructions.

4. Control and determination of the ovarian cycle stage for the collection of oocytes, zygotes, and embryos

1. Germinal vesicle (GV) oocyte collection from the ovaries
   1. Administer an intramuscular injection of 3 µL of 0.263 mg/mL cloprostenol (a synthetic PGF2α analog) diluted in 150 µL of saline¹⁴ (Figure 2) to reset the ovarian cycle at the end of the luteal phase (i.e., ≥10 days after the initiation of the luteal phase).
      NOTE: Making the diluted solution in a larger volume may be convenient as the diluted cloprostenol stays stable for, at least, several weeks at +4 °C.
   2. On the next day (day 1), confirm the initiation of the follicular phase by checking that the P4 level has dropped.
      NOTE: Cloprostenol injection has been reported to dramatically decrease P4 levels (usually <10 ng/mL) within 24 h³.
   3. From day 1, inject FSH (25 IU, intramuscular) once every 2 days for a total of 5x (days 1, 3, 5, 7, and 9). On day 10, inject hCG (75 IU, intramuscular) in the afternoon.
   4. Collect GVs from the ovaries on day 11 by follicular aspiration under anesthesia according to the literature^15,16.
      NOTE: Sometimes, ovulation occurs earlier than expected. Thus, it is recommended to check CG levels from day 8. If the CG test is positive, perform GV collection on this day.
2. Collection of metaphase II (MII) oocytes, zygotes, and early embryos
   1. Reset the ovarian cycle using cloprostenol as described in step 4.1.1.
   2. On the next day (day 1), confirm the initiation of the follicular phase by checking that the P4 level has dropped.
   3. For the collection of zygotes and embryos, house female marmosets together with male marmosets for mating from day 6.
   4. From day 7, check the blood P4/E2 levels and urine CG levels of the females. Detection of CG is an indication of ovulation within a few days (usually the next day). On the day that P4 levels increase and E2 levels decrease compared with the previous day, collect MII oocytes or embryonic day 0 (E0) zygotes from the oviducts^17,18.
   5. For embryo collection, as described in the literature, perform flushing either from the oviducts (E1-E3, 1-8 cells)^17,18 or the uterus (E5-E10, 8 cells-blastocyst)^19,20,21,22 at the appropriate time point, depending on the targeted stage.

Details related to the animals used in this study are listed in Table 1.

Anatomical analyses of the femoral vein
Anatomical analyses of the femoral vein were performed using a 2-year-old male common marmoset (I 7713M) undergoing euthanasia. The femoral veins and arteries are located in the femoral triangle. The femoral triangle is formed at the boundaries between the abdominal wall and thigh muscles (Figure 1B-D). At the base of the thigh, a large vein runs through the center of the inverted triangle, and an artery runs in parallel outside of the vein. In the lower region, the veins and arteries become thinner and overlap, with the arteries positioned on top of the veins (Figure 1D).

Venous blood should be drawn because an arterial injury can cause femoral artery hematoma, which may lead to cardiovascular shock when hemorrhage is severe¹³. Although blood can be drawn from any part of the vein in the triangle and its distal area, venipuncture from the proximal site of the femoral triangle is recommended because of the large size of the vein and its seemingly little overlap with the artery. Additionally, the vein in the proximal site is superficial, allowing for easy location using a needle. Because it pulsates, the artery in the femoral triangle is sometimes identified visually or by palpation, and the vein runs just medial to it. Thus, the observation of artery pulsation helps to predict the location of the vein.

Furthermore, a blue coloration indicative of the vein is usually observed under the skin at the top of the triangle (Figure 1B,C). However, lymph nodes in the triangle are often located close to the vein and show a dark blue color, so their appearance is similar. Fortunately, they can be distinguished by their mobility: the vein is stationary, and lymph nodes are movable. Thus, the pulsation of the artery and the blue color of the vein are two major clues used to locate the vein, although it may be necessary to shave the hair to visualize them.

Determination of the stage in the ovarian cycle
P4 and E2 levels were monitored in a 3-year-old female marmoset (I 6751F) for 38 days using the protocol described here. During this period, blood sampling and hormone measurements were performed every few days (Table 2).

Luteal phase (days 1-10)
The start day of the measurement was set as day 1. Based on the P4 level (21 ng/mL), the animal was likely in the luteal phase. A high P4 level was observed from days 1 to 10 (≥21 ng/mL), suggesting the luteal phase. On day 12, it dropped sharply to 4 ng/mL. This significant decrease indicated the transition from the luteal to the follicular phase. A decrease in E2 level from day 10 (241 ng/mL) to 12 (189 ng/mL) also supported this transition.

Follicular phase (days 12-22)
After transitioning to the follicular phase, the P4 level remained low (4-6 ng/mL) until day 19 and then slightly increased from day 19 to 24 (day 19, 6 ng/mL; day 22, 8 ng/mL; day 24, 9 ng/mL). In contrast, the E2 level significantly increased from day 19 (94 ng/mL) to 22 (322 ng/mL) and then decreased from day 22 to 24 (158 ng/mL). Based on the increased P4 and decreased E2 levels, ovulation was predicted to have occurred between days 22 and 24, transitioning from the follicular to the luteal phase.

Luteal phase (days 24-36)
After ovulation, the P4 level remained high until day 36 and then dropped to 3 ng/mL on day 38. Thus, it is likely that the transition from the luteal to the follicular phase occurred between days 36-38. Consistent with this transition, the E2 level decreased during this period (day 36, 2517 ng/mL; day 38, 73 ng/mL).

An additional five marmosets were monitored to investigate the duration of the follicular and luteal phases. The results showed an average duration of 11.58 days (n = 6 from four marmosets) and 16.8 days (n = 5 from three marmosets) for the follicular and luteal phases, respectively (Table 3).

Prediction and determination of the timing of ovulation
To examine the relationship between urine CG level and the ovulation date, seven marmosets (No. 1-7) were prepared. Cloprostenol was injected to reset the ovulatory cycle (day 0). Then, monitoring of blood P4/E2 levels and urine CG levels was performed from day 7. Urine was collected immediately after lighting. Blood sampling was essentially performed in the morning. It was assumed that the ovulation occurs when the E2 level was largely dropped compared with that in the previous day as reported¹⁷. On that day, the follicular rupture was indeed observed on the surface of the ovaries, and the presence of zygotes/oocytes in the oviducts (Table 4).

CG was first detected during days 7 to 11 (day 7, N = 1; day 8, N = 2; day 9, N = 1; day 10, N = 2; day 11, N = 1) (Table 4). The large drop in E2 levels (indication of the ovulation) was observed 0-3 days after the first CG signal. The duration from the first CG detection to the E2 decrease seemed to be large when the first CG detection was earlier (Figure 3A). For example, one animal (No. 1) showed a drop in E2 signal 3 days after the first CG detection on day 7 (Table 4). In contrast, the co-occurrence of the first CG detection and the drop of E2 signal was observed in one animal (No. 2) on day 10. Thus, although the first CG detection and ovulation were observed on the same day in one of the five animals, ovulation occurred within a few days after the first detection of CG.

The immunochromatographic test kit for marmoset CG is designed to use urine for testing. Examination of the CG level usually accompanies the determination of P4/E2 levels using blood plasma. It will be helpful if blood plasma, instead of urine, can be used for the CG test. To test this, the blood plasma that was left after the P4/E2 level measurement in the above experiments was examined. Blood plasma and urine were collected from the four animals on either of the days of the above experiments (the days of examination were indicated by double asterisks [**] in Table 4). Using urine, two of the four animals showed positive results (scores 5 and 3), and the other two showed negative results (score 1). Blood plasma showed essentially the same results as urine (Figure 3B). The stronger signal was obtained by using blood plasma. Therefore, when using blood plasma, the judgment should be made before 10 min, which is set for urine.

Figure 1: Blood collection from marmosets. (A) A restrainer is used for blood collection. (B) Marmoset upper thigh. (C) Femoral triangle and blood vessels. The femoral artery in the triangle is often visible and shows pulsation. The blue color of the femoral vein is sometimes visible in the proximal area of the triangle (indicated as the phlebotomy site). Lymph nodes also show a blue color. However, lymph nodes are movable as lymph nodes are attached to the skin. (D) Anatomical view of the thigh of the euthanized animal. The artery, vein, and phlebotomy site are indicated. The same animal is shown in B-D. (E) Blood collection using a restrainer. Holding position of the marmoset's legs and blood being drawn are shown. (F) Urine collection from a marmoset. Please click here to view a larger version of this figure.

Figure 2: Typical patterns of P4, E2, and CG levels during ovarian cycle in marmosets. The time points of FSH, hCG, and PGF2α injections are indicated. Dashed lines show the expected hormonal pattern after PGF2α injection. Abbreviations: P4 = progesterone; E2 = estradiol; CG = chorionic gonadotropin; FSH = follicle-stimulating hormone; hCG = human chorionic gonadotropin; PGF2α = prostaglandin F2α. Please click here to view a larger version of this figure.

Figure 3: Determination of CG level to predict the occurrence of ovulation. (A) The possible relationship between the first day of CG detection (score > 1) and duration until ovulation (E2 drop). (B) Blood plasma can be used for immunochromatographic CG tests. A representative result of each score (top). The score was determined 10 min after the sample loading. Score 2 represents no band within 5 min but the appearance of a band within 10 min. Blood collection was performed soon after the collection of urine. Four marmosets (No. 1, 2, 3, 5 in Table S4) were examined. Samples used here are indicated as double asterisks in Table 4. Blood plasma was diluted to 50% by dilution buffer used for E2 measurement. Please click here to view a larger version of this figure.

Table 1: Animals used in this study. Please click here to download this Table.

Table 2: Results of P4 and E2 measurements for 38 days in one marmoset.Please click here to download this Table.

Table 3: Duration of follicular and luteal phases. The phase was determined based on the P4 level (follicular phase P4 ≤ 8, luteal phase P4 > 8). When phase change was observed between the measurements, the midpoint between the measuring dates was determined to be the change point. The duration between the two change points was regarded as follicular or luteal phases. To ensure the measurement of the duration of a single phase, the data with a long (≥7 days) interval or twice of ≥6 day intervals in the same phase, including when spanning the two phases, were not used for the analyses. Only when two measurements were conducted in the same phase, the data were employed.Please click here to download this Table.

Table 4: Determination and prediction of ovulatory days. The traces of ovulation were considered to be present if bleeding sites or rupture sites were observed in ovaries. Oocyte/zygote indicates the stages of oocyte/zygote obtained from oviducts on the day of E2 drop.Please click here to download this Table.

Locating the vein is the most critical step in blood collection. Based on anatomical observations, this protocol introduces the proximal area in the femoral triangle as an easy site for blood collection in marmosets. Using this area, blood sampling from a large vein can be easily performed. However, even using this protocol, injury to an artery sometimes occurs. When injuring an artery, complete stopping of bleeding by applying pressure for >5 min is suggested to prevent hematoma. In addition, while applying pressure, cooling the punctate site using an ice cube is also effective. Hematomas can be treated with heparin (e.g., Tensolvet 5.000 I.E. gel). Most hematomas disappear after 3-4 consecutive days of treatment with heparin and 0.05 mL of meloxicam (0.5 mg/mL, p.o.). The artery runs laterally to the vein in the proximal area of the thigh. In the distal area, the vein and the artery seemed closer to each other and overlapped to a greater extent, with the artery located ventral to the vein. Based on this positional relationship, blood sampling from the proximal area of the thigh likely has a decreased risk of arterial injury.

In addition, the femoral vein in the proximal region is thick and located superficially, allowing for easy location using a needle. However, blood sampling from the distal area has the advantage of leaving the proximal sites intact, allowing for immediate repeated venipuncture from the proximal area of the same leg, even if the vein is damaged during blood sampling. This is because the distal area of the damaged site is usually unavailable for immediate venipuncture. However, when blood collection is unsuccessful, repeated sampling can be performed from the proximal site on the other leg.

The volume of circulating blood in marmosets is ~25 mL (70 mL/kg)²³. A 350 g marmoset requires 1 week to recover from 2 mL blood sampling, equivalent to 7.5% of the total blood volume⁶. From the femoral veins, 500-700 µL of blood can be collected without difficulty. When repeated sampling is needed, collecting smaller amounts of blood and the use of a dilution buffer for the automatic analyzer is recommended.

Although anesthesia is generally not used for blood sampling, for beginners, blood sampling from anesthetized animals may be much easier than from awake animals. However, caution is warranted when using anesthesia for blood sampling because it was reported that alfaxalone resulted in unexpectedly higher levels of P4²⁴. This was caused by the cross-reactivity of anti-P4 antibody to alfaxalone, which is a P4 derivative.

Structures of P4 and E2 (steroid hormones) are identical between humans and marmosets. Commercial ELISA kits for human P4 and E2 are, therefore, available for marmoset P4 and E2. Outsourcing services for human blood hormone measurement are also available. By contrast, there are slight differences in amino acid sequences for polypeptide hormones, such as FSH, CG, and Inhibin. For these polypeptide hormones, if a kit can be used for both humans and other evolutionary distant species, such as mice, it will work for marmosets as well. For Inhibin B measurement, a Human/Mouse/Rat Inhibin B (Beta B subunit) Enzyme Immunoassay Kit can be used.

An increase in the P4 level is a hallmark of ovulation (transition from the follicular to the luteal phase). Although variations among individuals make it difficult to pinpoint the date of ovulation from the P4 level alone, a P4 level of >8 ng/mL can be used as a rough indicator of the occurrence of ovulation. A more reliable indicator of the ovulation date is a decrease in the E2 level^3,17. Ovulated oocytes/zygotes were indeed observed in the oviducts on the day of E2 decrease (Table 4). Thus, the measurement of E2 enables us to pinpoint the exact ovulation date. Besides the hormonal levels, information on the number of days after PGF2α administration is a predictor of the day of ovulation (Table 4). Similarly, combinatory use of P4 and E2 levels helps to identify the timing of the transition to the follicular phase, when P4 levels decrease dramatically and E2 levels are at their lowest.

In marmosets, CG, instead of LH, is released from the pituitary gland to induce ovulation. Additionally, CG is excreted from trophoblast cells when embryos attach to and implant in the endometrium. Because marmosets lack a functional LH gene, CG, instead of LH, is released from the pituitary gland to stimulate ovulation⁵. A study reported that E2 and CG peaks, indicative of ovulation, are observed on the same day¹². Another study reported that, on average, E2 peaks (8.6 days after PGF2α administration) precede CG peaks (9.3 days after PGF2α administration)²⁵, indicating that ovulation may occur before reaching the CG peaks. CG, unlike E2, is pulse-released just before its peak. Pulse-release of CG starts, on average, ~1 day before its peak. However, a large variation in duration from the first detection of the CG level to the drop in the E2 level was observed (Figure 3A and Table 3). Additionally, CG is reported to be first detected in both morning and afternoon samples²⁵.

To our knowledge, there is no report of ovulation in marmosets occurring at a particular time of day. In addition, marmosets usually release two or three oocytes at once, so there may be a certain period over which all oocytes are released. Since increased CG levels occur before ovulation, CG measurement enables the prediction of the ovulation date in advance. Such information is particularly useful for the collection of MII oocytes or day 0 embryos on the day of ovulation. Thus, a combinatorial measurement of blood P4/E2 levels and CG levels is useful (Figure 3B). In most primate species, including humans, ovulation does not occur for a while after delivery. However, postpartum ovulation occurs in marmosets ~10 days after delivery. Consequently, the next delivery is often observed ~155 days after the previous delivery. This sequential pregnancy, together with the delivery of twins, results in a high level of fecundity in marmosets.

Hormonal fluctuations vary among individuals, and some do not have a constant cycle. Some female marmosets show constant low P4/E2 levels, which indicates a lack of ovulation. The lack of an ovulatory cycle can be attributed to immaturity, health problems, stress, or ovulation inhibition by dominant reproductive females^26,27. Three of the 21 marmosets (>2-year-old) measuring hormonal levels showed the lack of a regular ovulatory cycle in the author's facility. Understanding the causes and developing appropriate treatments will enable the more effective use of animals in experiments. Individuals with a prolonged luteal phase of ~1 month and an associated high P4 level were observed². In the author's facility, a prolonged luteal phase is repeated in the same individuals. Two of the 21 marmosets showed this abnormality. One of the two animals was single-housed, excluding the possibility that the animal was pregnant during the prolonged luteal phase. The other is paired with a presumably infertile male by radiation treatment, and therefore she was unlikely pregnant during the prolonged luteal phase. Even in a prolonged luteal phase, PGF2α administration lowers the P4 level and resets the ovarian cycle. Measuring sex hormones and understanding the ovulation cycle status helps the effective use of animals in experiments and breeding.

Blood sampling from individuals is used not only for ovulation date estimation but also for serological tests for diagnostic purposes. Similarly, the measurement of hormone levels is used not only for estimating the day of ovulation but also for studying social interaction, child development, and diseases. In the future, for making genetically modified marmosets, developing ovaries and testes from iPS cells is expected. Appropriate hormonal levels should be important for normal development. The protocol described here will be useful for the measurement and control of sexual hormones for making iPS cell-derived gonads in marmosets.