Traits Of Polycystic Ovary Syndrome In Prenatally Androgenized Female Sprague-Dawley Rats

ForewordPolycystic ovarian syndrome (PCOS), mainly featuring the androgen excess and the chronic anovulation, is one of the most common endocrine disorder diseases among the women of childbearing ages. The incidence rate of it accounts for 5-10% among the reproductive age women and 75-80% among the anovulatory women. The unity of the diagnostic criteria about PCOS is first seen in 1935, Stein and Leventhal described it as non-ovulation-related syndrome featuring amenorrhea, hypertrichiasis and polycystic ovary. In 1990 National Institute Health (NIH) meeting of US defined its diagnostic criteria as "a clinical or biochemical hyperandrogenism performance, and chronic anovulation with the exception of other diseases". But after years of clinical observations, most Ultrasound find that many PCOS patients have polycystic ovarian. Therefore, the diagnostic criteria have been controversial. In 2003 in Rotterdam, the Netherlands defined the new diagnostic criteria for PCOS. The morphological changes of the ovary-"polycystic ovary" is as one of its diagnostic criteria. Rotterdam diagnostic criteria: (1) Oligo-and/or anovulation; (2) Clinical and/or biochemical signs of hyperandrogenism; (3) Polycystic ovaries and exclusion of other aetiologies (congenital adrenal hyperplasias,androgen-secreting tumours, Cushing's syndrome). The current standards about AES in 2006: (1) hypertrichiasis and/or hyperandrogenism; (2) Oligo- and/or anovulation and / or polycystic ovary (3) exclusion of other aetiologies, such as CAH, Cushing's syndrome, High Prolactinemia, serious insulin resistance syndrome, androgen secretion of tumor, thyroid dysfunction, and so on.There are also other signs and symptoms associated with PCOS: reproductive endocrine aspects: (1) high LH; (2) excessive secretion of adrenal androgen; (3) ovarian excessive response to gonadotropin stimulation; (4) IVF High abortion rate; (5) endometrial hyperplasia and endometrial cancer; (6) gestational diabetes. Metabolism: (1) insulin resistance and hyperinsulinemia; (2) lower glucose tolerance; (3) Type 2 diabetes; (4) obesity; (5) damage to the pancreas respond to glucose; (6) hyperlipidemia. In addition, cardiovascular disease, sleep apnea, acne, the increased incidence of chronic inflammation.At present the reasons for PCOS is still not clear, may be a genetic and environmental factors to determine it a heterogeneous disease. There is only palliative treatment. It can not get at the root. To further explore its causes and pathogenesis of PCOS play a great role in the diagnosis and treatment. However, Human limitations about PCOS research, especially in the mechanism, so the corresponding animal studies have become necessary. The model such as exogenous androgen created model, estrogen created model, sustained light created model, are able to reflect some PCOS symptoms and signs, but can not fully reflect PCOS endocrine changes and changes in local sexual gland. But more importantly, they can not explain its etiology too. How to do a animal research better to solve the problem is a questionIn order to address these conflicts, scientists made a large number of studies, the researchers found: fetal period, tissues and organs of the body will experience the sensitive stage of development. This sensitive stage may be related to the rapid secession of the cells. And at this sensitive stage, some stimulation or injury, will lead to life-long impact. And the recent animal studies show that, PCOS may be relevant. May be in the fetal period, the cycle of excessive androgen stimulation has an impact on fetus, leading to the formation of adult PCOS. Abbott study found that if rhesus monkeys exposure to excessive androgen during pregnancy, the adult signs and symptoms is similar to patients with PCOS. It was the first to observe the PCOS origin, which confirmed "fetal origins of adult disease hypothesis".For these reasons, we want to use SD rats to do experiments. Prenatal hormone exposures were accomplished by treatment of pregnant dams from Embryonic Day 16 to Embryonic Day 19 with daily injections (i.p., s.c.) of either free T or DHT, or oil injection solution. The drug dose followed the researchers of the previous relevant literature. This dose is capable of partially masculinizing the anal-genital distance and sex behavior. The pregnancy cycle about rat is 21-22 days (normal gestation period of 21 days). We want to view its symptoms and signs more similar to PCOS patients and to further explore and analyse the reasons for PCOS at the same time.ObjectiveUnknown etiology about PCOS, it is more improtant to further explore its causes and pathogenesis for treatment. However, there are limitations on PCOS about the human body study, especially in the pathogenesis of respect. For example, we can not removed ovary and get hypothalamus specimens, and so on. Many animal models reflect PCOS symptoms and signs from different aspects, but it is important to note the reasons. How to do a animal research better to solve the problem.is a question. For these reasons, the main topic is to explore a new model ofPCOS.1. Prenatal hormone exposures were accomplished by treatment of pregnant dams from Embryonic Day 16 to Embryonic Day 19 with daily injections (i.p., s.c.) of either free T or DHT, or oil injection solution. Offspring of each group is given EB after ovariectomy. PgR mRNA expression is analysed and understand the change about their secretion of hormones from molecular biology and the reasons for the change.2. We observed the genital changes after the birth and understand androgen influence about the female.3. Observation of the estrous cycle and hormone secretion way change of prenatal androgen excess rats, compared with PCOS patients with endocrine changes in the menstrual cycle.4. We research the characteristics of glucose metabolism about prenatal androgen excess and normal rats, to study the changes of the rat metabolism.Methods1. Female and male rats were housed in the ratio of 3:1 or 4:1 per cage and in the next morning the vaginal smears were checked to observe the presence of vaginal plug which indicated positive mating. This day was considered day one (D1) of pregnancy. Prenatal hormone exposures were accomplished by treatment of pregnant dams from Embryonic Day 16 to Embryonic Day 19 with daily injections (i.p., s.c.) of either free T or DHT, or Oil for Injection solution. At 11:00 am to 11:30 am about the 81st day (at the age of 70 future period, physiological structure is mature), the control group is rapidly injected estradiol benzoate (estradiol benzoate, EB) 30ug in diestrus period after the surgical excision of ovarian, the experimental group is done at the same. The next day 12:00 rats were sacrificed quickly by decapitatation, the preoptic area (preoptic area, POA) was preserved in liquid nitrogen, followed by RT-PCR test of its PgR expression, to understand the reasons for the change in hormone secretion from molecular biology.2. The No 2, No 22, No 60 when offspring was born (postnatal day 2, PND2, PND22, PND60) anal genital distance was measured (the distance between the base of the genital papilla and the rostral end of the anal opening, namely anogenital distance, AGD) and the 13th check nipple situation.3. (1) At 11am to 11:30am in the day 60-70, control group of rats in diestrus stage, the experimental group on the same date (without the vagina, cann't do vaginal smear), injected sodium pentobarbital anesthesia (40mg/kg, ip), blood sample drawing from the cavainferior using sterile syringes, the hormone secretion was observed and analysed; (2) The control group in diestrus period, the experimental group at the same time, ovary is surgically excised, 12:00 to 15:00 after seven days, blood sample drawing from orbital vein every 10 minutes, a total of three hours, serum LH is determined by RIA, understanding LH secretion changes in frequency and amplitude.4. After 70 days, all rats began fasting at 20:00, with administration of 50 percent glucose by 3 g glucose/kg body weight dose at 8:00 the next day. In 0, 30, 60, 120 min after the irrigation of sugar, blood sample drawed from tail. Blood glucose determined by Blood Glucose Meter, insulin-free detected by RIA.Results1. EB stimulation after ovariectomized, PgR expression of comparison:the expression of PgR in the control group(1.2112±0.2355)higher than the expression of the experimental groups(0.5017±0.3234)and(0.4123±0.2876),P <0.05.2. (1) AGD after the birth of offspring about groups. The control group compared with the experimental groups: PND2 (followed by:1.4±0.1vs1.6±0.1,1.4±0.1vs1.7±0.1, both P<0.01); PND22(followed by 10.5±0.4 vs 14.2±1.1,10.5±0.4 vs 14.9±0.5, P <0.01); PND60(followed by 15.1±0.5 vs 19.1±0.4, 15.1±0.5 vs 19.0±0.3, P<0.01). However, comparison between the experimental group:PND2 (followed by:1.6±0.1 vs 1.7±0.1, P>0.05); PND22(followed by 14.2±1.1 vs 14.9±0.5, P>0.05); PND60 (followed by 19.1±0.4 vs 19.0±0.3, P> 0.05). (2) Aeola inspection after the birth of offspring about groups on PND13. The control group and experimental groups: normal areola few (11.9±0.3 vs 1.5±0.5, 11.9±0.3 vs 1.3±0.5, P<0.05), the control group and experimental group differences were statistically significant. Experimental group (1.5±0.5 vs 1.3±0.5, P>0.05), not statistically significant. The total number of areola (12±0.0 vs 2.8±0.8, 12±0.0 vs 2.4±0.7, P<0.05). There were statistically significant about the control group and experimental groups, not statistically significant between the experimental groups (2.8±0.8 vs 2.4±0.7, P>0.05,),.3. (1) Hormone determination. The control group with the experimental groups (T group, DHT group) comparison respectively, followed by E2 (15.3±0.5 vs 23.0±1.2, 15.3±0.5 vs 23.0±1.2, P<0.05)(ng/ml); P(15.3±0.7 vs 24.7±0.6, 15.3±0.7 vs 24.7±2.1, P<0.05)(ng/ml); LH(0.44±0.07 vs 0.77±0.07, 0.44±0.07 vs 0.76±0.07, P<0.05)(ng/ml), there are statistical significance.T (0.019±0.007 vs 0.021±0.008, 0.019±0.007 vs 0.024±0.008, P>0.05) (ng/ml), there is not statistical significance between the control group and experimental groups. Experimental group were no statistical significance too. (2) LH secretion of frequency and magnitude of rats in each group. Observed that: the experimental group (T) rats compared with control group, the increasing frequency, greater magnitude. but there is no difference in DHA group because of the experiment.reasons4. OGTT results of rats about each group. Fasting plasma glucose (4.4±0.2 vs 4.4±0.3, 4.4±0.2 vs 4.3±0.2, P>0.05)(mmol/L); 1h glucose(7.43±0.21 vs 7.45±0.22, 7.43±0.21 vs 7.31±0.34, P>0.05) (mmol/L); 2h blood glucose (6.18±0.14 vs 6.18±0.14, 6.18±0.14 vs 6.06±0.20, P>0.05) (mmol/L). Three sets of fasting insulin, respectively (13.9±1.0 vs 14.2±0.6, 13.9±1.0 vs 14.2±1.0, P>0.05) (mU/L); 2h insulin (12.0±0.7 vs 11.5±0.8, 12.0±0.7 vs 11.9±1.0, P>0.05)(mU/L).Conclusions1. E2 to normal female rats can induce the expression of PgR mRNA and LH peak. In androgen excess rats during pregnancy, the expression of PgR mRNA is reduced, reduced PgR lead to reduced GnRH secretion of the hypothalamus, thus do not have LH peak. PCOS patients have abnormal secretion of LH, reduced about the ovulation This may be the reason about PCOS .2. AGD increased significantly, the number of nipples significantly reduced too, It also showed female pseudohermaphroditism. These are similar to the some of human disease. Using of androgen and progesterone at pregnancy can lead to female pseudohermaphroditism.3. Prenated androgen excess rat model also has high LH secretion and estrous cycle change(cycle extension). We can see LH/FSH ratio>2, even 3, menstrual disorders, Oligo-and/or anovulation in PCOS patients. We speculate excessive androgen during pregnancy may lead these changes.4. PCOS patients have lower glucose tolerance, increased risk of diabetes. Prenatal androgen excess rats have no abnormal changes in metabolism, it may be related to different species.