PAG and progesterone PAG and progesterone profiles in IVF pregnancy profiles in IVF pregnancy

after hormonal stimulation after hormonal stimulation and ovum-pick-up method and ovum-pick-up method

in cowsin cows

A.Tomašković, A.Tomašković, N. Prvanović,N. Prvanović, J. F. Beckers, J. F. Beckers, J. Sulon, M. Cergolj , I. Getz, J. Grizelj,J. Sulon, M. Cergolj , I. Getz, J. Grizelj,

M. Samardžija , T. KaradjoleM. Samardžija , T. Karadjole

Our goal was:Our goal was: to determine the influence of using cows to determine the influence of using cows

for two different procedures in assisted for two different procedures in assisted reproduction on later fertility and reproduction on later fertility and possibility to conceive by measuring levels possibility to conceive by measuring levels of progesterone and PAG and preforming of progesterone and PAG and preforming transrectal ultrasound examinationtransrectal ultrasound examination

to monitor foetal development using to monitor foetal development using ultrasound and measuring the foetusesultrasound and measuring the foetuses and making PAG curveand making PAG curve

Introduction:Introduction: we can determine pregnancy by we can determine pregnancy by

presence of corpus luteum on the presence of corpus luteum on the ovarium and consequently high ovarium and consequently high concentration of progesterone in concentration of progesterone in peripheral circulation and peripheral circulation and according to ultrasound findingaccording to ultrasound finding

presence of alive, vital embryo in presence of alive, vital embryo in development can be proved only by development can be proved only by determination of determination of maternal maternal recognition of pregnancyrecognition of pregnancy

on the clinical point of view, the on the clinical point of view, the monitoring of P4, bPAG or BPSPB monitoring of P4, bPAG or BPSPB may help to detect earlier placental may help to detect earlier placental abnormalities and embryonic or abnormalities and embryonic or foetal mortalityfoetal mortality

after ex-vivo embryo transfer, the after ex-vivo embryo transfer, the occurence of embryo defect is very occurence of embryo defect is very low, close to those obtained after low, close to those obtained after natural mating or artificial natural mating or artificial insemination.insemination.

Material and methods:Material and methods:

Blood samples of 18 cows divided in three groups Blood samples of 18 cows divided in three groups 1.1. 6 IVF pregnancy cows6 IVF pregnancy cows after hormonal stimulation and after hormonal stimulation and

ovum-pick-up method, ovum-pick-up method, 2.2. 6 pregnant cows after AI 6 pregnant cows after AI 3.3. 6 nonpregnant cows6 nonpregnant cows

Blood samples 21 days after fertilization till 40 days Blood samples 21 days after fertilization till 40 days for pregnancy diagnosis. for pregnancy diagnosis.

All cows in our experiment had no history of All cows in our experiment had no history of reproductive disturbances or difficult reproductive disturbances or difficult parturations and were3-6 years old.parturations and were3-6 years old.

We analysed:We analysed:

RIARIA ( (progesterone and bPAGprogesterone and bPAG)) level of progesterone at day 21 level of progesterone at day 21

level of bPAG at day 21., 24., 30., 35. and 40 - level of bPAG at day 21., 24., 30., 35. and 40 - specific curve for each cowspecific curve for each cow

data about cowsdata about cows and esspecially and esspecially service service intervalinterval

rectal palpationrectal palpation and and transrectal real-time transrectal real-time ultrasoundultrasound with linear probe few times after with linear probe few times after day 20 and than at day 40, 50, and pregnant day 20 and than at day 40, 50, and pregnant cows at aproximatly day 90, 120 and 150.cows at aproximatly day 90, 120 and 150.

Cows in 3 groups were:Cows in 3 groups were: Cows carring IVF pregnancyCows carring IVF pregnancy in present in present

investigation ware previously used as OPU/IVF investigation ware previously used as OPU/IVF donors after hormonal stimulation with FSHdonors after hormonal stimulation with FSH

6 cows that stayed pregnant after AI6 cows that stayed pregnant after AI 6 nonpregnant cows6 nonpregnant cows were not inseminated but were not inseminated but

are confirmed to be healthy and had ovarial are confirmed to be healthy and had ovarial cyclical activity using rectal palpation and cyclical activity using rectal palpation and ultrasound examination at days 20, 40. and 50 ultrasound examination at days 20, 40. and 50 after the parturationafter the parturation

OPU/IVF donors OPU/IVF donors procedure:procedure:

Cows carring IVF pregnancy in present Cows carring IVF pregnancy in present investigation ware previously used investigation ware previously used as OPU/IVF donors after:as OPU/IVF donors after:

hormonal stimulation with FSH hormonal stimulation with FSH during 3 days with equal doses of during 3 days with equal doses of Folltropin®-V (Vetrepharm Inc., Folltropin®-V (Vetrepharm Inc., London, Canada, a total dose: 300 London, Canada, a total dose: 300 mg NIH-FSH-P1)mg NIH-FSH-P1)

OPU was performed 48 hours after OPU was performed 48 hours after the last FSH injectionthe last FSH injection

Procedure was repeated 3 times Procedure was repeated 3 times

during 8 months in intervals of 60-during 8 months in intervals of 60-90 days90 days

2 months after the last ovum pick-2 months after the last ovum pick-up session transfer of IVF embryos up session transfer of IVF embryos was performed in these cowswas performed in these cows

Results:Results: cows in control group conceived after cows in control group conceived after

A.I.A.I.

6-2=4 pregnant cows6-2=4 pregnant cows group of cows with IVF pregnancy group of cows with IVF pregnancy

after hormonal stimulation and ovum-after hormonal stimulation and ovum-pick-up methodpick-up method

4-3-1 pregnant cow4-3-1 pregnant cow all other were confirmed to be all other were confirmed to be

nonpregnantnonpregnant

0 pregnant cows0 pregnant cows

CowsCows Pregnant at 1. Pregnant at 1. control-after day control-after day

20.20.

Pregnant at 2. Pregnant at 2. control-day 40.control-day 40.

Pregnant at 3. Pregnant at 3. control-day 50.control-day 50.

AI cowsAI cows 66 44 44

IVF cowsIVF cows 44 33 11

Nonpregnant Nonpregnant cowscows

00 00 00

level of progesterone level of progesterone

below 2 ng/ml nonpregnant cow below 2 ng/ml nonpregnant cow

2-10 ng/ml pregnant cow2-10 ng/ml pregnant cow

level of bPAG- specific individual curvelevel of bPAG- specific individual curve

increase of bPAG pregnant cows increase of bPAG pregnant cows decrease of bPAG or irregular curve nonpregnant decrease of bPAG or irregular curve nonpregnant cows cows

service intervalservice interval- level of bPAG decreases in - level of bPAG decreases in maternal blood 70 days after parturation or maternal blood 70 days after parturation or embryonal / foetal death. embryonal / foetal death.

We compared all numbered data We compared all numbered data ((progesterone level, bPAG curve, service progesterone level, bPAG curve, service intervalinterval) with results of rectal and ultrasound ) with results of rectal and ultrasound examination examination

We concluded that repeated rectal and We concluded that repeated rectal and ultrasound examination gave the same results ultrasound examination gave the same results as progesterone checking and bPAG curve. as progesterone checking and bPAG curve.

All cows suspected to be All cows suspected to be nonpregnant were confirmed to nonpregnant were confirmed to be nonpregnant and all cows be nonpregnant and all cows suspected to be pregnant were suspected to be pregnant were confirmed to be pregnant, by all confirmed to be pregnant, by all used methodsused methods

Conclusions:Conclusions:IVF pregnancy after hormonal stimulation and ovum-pick-up IVF pregnancy after hormonal stimulation and ovum-pick-up

method in cows carries high risk of embrional death but it method in cows carries high risk of embrional death but it is very complex question and we definitely need to preform is very complex question and we definitely need to preform more research wich includes bigger number of animals and more research wich includes bigger number of animals and more parameters more parameters

We also need closer monitoring of each cow, frequently We also need closer monitoring of each cow, frequently preformed rectal and ultrasound examination, frequent preformed rectal and ultrasound examination, frequent blood sampling during whole pregnancy and making bPAG blood sampling during whole pregnancy and making bPAG curve, which together with progesterone level can be curve, which together with progesterone level can be valuable tool in detecting and diagnostics of early valuable tool in detecting and diagnostics of early embrionic death embrionic death

Hopefully all together can help to improve success of all Hopefully all together can help to improve success of all methods used in assisted reproduction.methods used in assisted reproduction.

Thank You for attention and time!