Timing of hormone therapy and its association with cardiovascular risk and metabolic parameters in 4-vinylcyclohexene diepoxide-induced primary ovarian insufficiency mouse model

Figures & data

Figure 1. Study design and VCD treatment results.

A. Flow-chart visualization of the study group classification.

IP, intra-peritoneal; VCD, 4-vinylcyclohexene diepoxide with 160mg/kg; ND, normal diet; E2, estradiol with 0.2mg/kg; NT, no estradiol treatment; T1, treatment group 1 with delayed estradiol treatment for 3 weeks; T2, treatment group 2 with on-time, continuous estradiol treatment for 6 weeks; T3, treatment group 3 with on-time, short estradiol treatment for 3 weeks; PO, per oral; NS, normal saline 0.9%; HFD, high fat diet.

B. AMH and FSH levels after the completion of VCD treatment.

AMH, anti-Müllerian hormone; FSH, follicle-stimulating hormone; VCD, 4-vinylcyclohexene diepoxide. *P-value <.05 (vs. vehicle).

Table 1. Study design and group classification.

Group	Tx type and dose	Volume (ml/kg)	Tx duration (week)	Route of administration	Diet
Cont (n = 10)	VCD, 160 mg/kg	2.5	1^st to 2^nd	IP	ND
	NS, 0.9%	10	3^rd to 8^th	PO	ND
NT (n = 10)	VCD, 160 mg/kg	2.5	1^st to 2^nd	IP	ND
	NS, 0.9%	10	3^rd to 8^th	PO	HFD
T1 (n = 10)	VCD, 160 mg/kg	2.5	1^st to 2^nd	IP	ND
	NS, 0.9%	10	3^rd to 5^th	PO	HFD
	E2, 0.2 mg/kg	10	6^th to 8^th	PO	HFD
T2 (n = 10)	VCD, 160 mg/kg	2.5	1^st to 2^nd	IP	ND
	E2, 0.2 mg/kg	10	3^rd to 8^th	PO	HFD
T3 (n = 10)	VCD, 160 mg/kg	2.5	1^st to 2^nd	IP	ND
	E2, 0.2 mg/kg	10	3^rd to 5^th	PO	HFD
	NS, 0.9%	10	3^rd to 8^th	PO	HFD

Tx, treatment; Cont, control; VCD, 4-vinylcyclohexene diepoxide; NS, normal saline; IP, intra-peritoneal; PO, per oral; ND, normal diet; NT, no estradiol treatment; HFD, high fat diet; T1, treatment group 1 with delayed estradiol treatment for 3 weeks; E2, estradiol; T2, treatment group 2 with on-time estradiol treatment for 6 weeks; T3, treatment group 3 with on-time estradiol treatment for 3 weeks.

Figure 2. Effect of estradiol (E2) treatment on physiologic status of POI mice during the course of HT.

A. Body weight change pattern. B. Fasting blood glucose (FBG) level change pattern.

FBG, fasting blood glucose; Cont, control; NT, no estradiol treatment; T1, treatment group 1 with delayed estradiol treatment for 3 weeks; T2, treatment group 2 with on-time estradiol treatment for 6 weeks; T3, treatment group 3 with on-time estradiol treatment for 3 weeks. ^#P-value <0.001 (vs. NT); ^##P-value <0.01(vs. NT).

Figure 3. Effect of estradiol (E2) streatment on metabolic parameters in POI mice with different initiation time points and durations of HT.

A. Serum total cholesterol levels. B. Serum triglyceride levels. C. Serum insulin levels. D. Serum low-density lipoprotein-cholesterol levels.

Cont, control; NT, no estradiol treatment; T1, treatment group 1 with delayed estradiol treatment for 3 weeks; T2, treatment group 2 with on-time estradiol treatment for 6 weeks; T3, treatment group 3 with on-time estradiol treatment for 3 weeks; LDL-C, low-density lipoprotein-cholesterol. *P-value <0.001 (vs. Cont); **P-value <0.01 (vs. Cont); ^#P-value <0.001 (vs. NT); ^##P-value <0.005 (vs. NT); ^†P-value <0.005 (vs T2); ^‡P-value <0.001(vs T2).

Figure 4. Effect of estradiol (E2) treatment on aortic thickness (μm) with different initiation time points and durations of HT.

A. Transverse section of descending thoracic aorta of the study subjects. B. Comparison of aorta thickness among the study groups.

Cont, control; NT, no estradiol treatment; T1, treatment group 1 with delayed estradiol treatment for 3 weeks; T2, treatment group 2 with on-time estradiol treatment for 6 weeks; T3, treatment group 3 with on-time estradiol treatment for 3 weeks. *P-value <0.001 (vs. Cont); ^#P-value <0.005 (vs. NT).

Figure 5. Effect of estradiol (E2) treatment on serum pro-inflammatory cytokines in POI mice with different initiation time points and durations of HT.

A. Serum TNF-α. B. Serum IL-1β.

TNF-α, tumor necrosis factor-α; IL-1β, Interleukin-1β;Cont, control; NT, no estradiol treatment; T1, treatment group 1 with delayed estradiol treatment for 3 weeks; T2, treatment group 2 with on-time estradiol treatment for 6 weeks; T3, treatment group 3 with on-time estradiol treatment for 3 weeks. *P-value <0.001 (vs. Cont); ^#P-value <0.001 (vs. NT); ^##P-value <0.005 (vs. NT).

Table 2. Effect of estradiol (E2) treatment on organ weights in grams (g) of POI mice with different initiation time points and durations of HT.

Organ	Cont	NT	T1	T2	T3
Liver	1.30 ± 0.05	1.38 ± 0.06	1.03 ± 0.09	1.03 ± 0.13	1.09 ± 0.05
Heart	0.12 ± 0.02	0.11 ± 0.01	0.11 ± 0.01	0.11 ± 0.01	0.11 ± 0.01
Spleen	0.074 ± 0.004	0.076 ± 0.004	0.079 ± 0.003	0.078 ± 0.003	0.078 ± 0.003
Kidney	0.193 ± 0.009	0.182 ± 0.006	0.179 ± 0.009	0.176 ± 0.012	0.182 ± 0.010
Visceral fat	0.109 ± 0.028	0.510 ± 0.057*	0.400 ± 0.097^*, †	0.406 ± 0.079^*, †	0.515 ± 0.178*
Uterus	0.096 ± 0.014	0.091 ± 0.006	0.087 ± 0.008	0.090 ± 0.010	0.087 ± 0.010
Ovary	0.003 ± 0.001	0.003 ± 0.001	0.005 ± 0.001^#	0.005 ± 0.001^#	0.004 ± 0.001^#

Data presented as mean ± SD in grams. Cont, control; NT, no estradiol treatment; T1, treatment group 1 with delayed estradiol treatment for 3 weeks; T2, treatment group 2 with on-time estradiol treatment for 6 weeks; T3, treatment group 3 with on-time estradiol treatment for 3 weeks. *P-value <.001 (vs. Cont); ^#P-value <.001 (vs. NT); ^†P-value <.001 (NT vs. T1, T2).

Figure 6. Summary of the findings and evidence-based interpretations of HT in POI.

E, estrogen; HT, hormone therapy.