Vitamin D level is associated with increased clinical pregnancy rate and live birth rate in Chinese women undergoing IVF/ICSI-ET: a retrospective cohort study

doi:10.21203/rs.3.rs-1882224/v1

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Vitamin D level is associated with increased clinical pregnancy rate and live birth rate in Chinese women undergoing IVF/ICSI-ET: a retrospective cohort study

https://doi.org/10.21203/rs.3.rs-1882224/v1

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Research question

To explore the relationship between serum 25(OH)D level and Clinical Pregnancy Rate (CPR) as well as Live Birth Rate (LBR) in Chinese women receiving IVF/ICSI-ET.

Design

The data of 612 patients who underwent IVF/ICSI-ET in the Reproduction Center of the Second Affiliated Hospital of Wenzhou Medical University were retrospectively analyzed.

Results

None of the baseline characteristics of participants were significantly different between the two cohorts. The trend of 25(OH)D concentration was positively correlated with CPR and LBR. Moreover, our results indicated that the younger (Age: p<0.001 both in CPR and LBR) women with primary infertility (Infertility type: p=0.004 in LBR) were more likely to get a better pregnancy outcome under the same 25(OH)D concentration stages. As shown on heatmap plots, CPR and LBR were significantly increased for 25(OH)D concentrations above 30.00ng/ml and women younger than 30 years old. The adjusted binary logistic regression and RCS showed that there exists a nonlinear positive correlation between 25(OH)D concentration and pregnancy outcome (CPR and LBR) (nonlinear < 0.001, respectively). The women with a sufficient 25(OH)D concentration (30ng/ml) had 1.07 (clinical pregnancy) and 1.05 (live birth) times higher successful birth outcomes compared to women with an insufficient 25(OH)D concentration (25ng/ml). (OR_{25ng/ml,ref=30ng/ml}[95%CI] = 0.935[0.932-0.938] and 0.947[0.945-0.950],P<0.001, respectively)

Conclusion

In Chinese women receiving IVF/ICSI-ET, the serum level of 25(OH)D demonstrated a non-linear positive correlation with pregnancy outcome（CPR&LBR, with stronger correlations above 25 ng/ml and worse yields below 30 ng/ml. However, it could not yet be considered different in distinct ages.

Vitamin D is mainly synthesized through the UV-B synthesis reaction of skin exposed to sunshine^1,2. 25(OH)D deficiency is very common worldwide^3,4, and it is more obvious in Asians. According to the Chinese National Nutrition Surveillance (CNHS) ⁵, 25(OH)D deficiency is more frequent in women at the age of childbearing in China. This is probably attributed to sunlight time, intensity, and VDR gene polymorphisms, such as BsmI (Rs1544410) ^6,7.

Researchers have so far focused on the effect of 25(OH)D on bones. However, 25(OH)D is involved in numerous mechanisms in the field of reproductive medicine. In neonates, it promotes the development of bone, nervous system, and lung⁸. In pregnant women, it attributes to endometrial calcium homeostasis maintenance, immune regulation, it has an anti-inflammatory effect during pregnancy and it reduces the risk of rickets after birth^9–11. Moreover, research by Parikh et al. showed that 25(OH)D promotes the production of numerous hormones including estrogen, progesterone, estradiol, and insulin-like growth factor binding protein 1 (IGF-BP-1) ^12,13. Furthermore, Hahn et al. found that the 25(OH)D deficiency may be associated with insulin resistance, hyperandrogenemia polycystic ovary syndrome, and ovulation disorders^13–16.

Although the importance of 25(OH)D on fetal development and pregnancy is gradually being recognized, the effect of 25(OH)D deficiency on assisted reproductive outcomes remains unclear. The In Vitro Fertilization (IVF)/Intracytoplasmic Sperm Injection (ICSI)-EmbryoTransfer (ET) (IVF/ICSI-ET) is a traditional and common technique for assisting reproduction, but the existing studies on IVF/ICSI-ET have mainly focused on the non-Asian population. There is a lack of systematic studies on the pregnancy outcomes of Asians. Our study is thus studying the associations of the serum 25(OH)D levels in Chinese women who had received IVF/ICSI-ET with their clinical pregnancy outcomes.

Patients

The patients who underwent IVF/ICSI-ET in the Second Affiliated Hospital of Wenzhou Medical University from May 2018 to December 2019 were retrospectively analyzed. This study had been ratified by the institutional review committee of the Second Affiliated Hospital of Wenzhou Medical University. All patients were consulted extensively before obtaining standard informed consent and were divided into two cohorts according to their 25(OH)D concentrations (< 30ng/ml (560), ≥ 30ng/ml(52)) and four groups for further studies (G1:<20ng/ml(284), G2:20-25ng/ml(170), G3:25-30ng/ml(106), G4:≥30ng/ml(52)).

Serum 25(OH)D measurement

All patients underwent routine serum 25(OH)D concentration measurements by liquid chromatography-mass spectrometry (LC-MS) before IVF/ICSI-ET.

IVF/ICSI-ET procedure

The long-acting protocol for ovarian stimulation was as follows: A total of 3.75 mg of gonadotropin-releasing hormone agonist (GnRH-a) was administered starting from Days 1–3 of the menstrual cycle. Inclusion criteria were examined after 35–38 days, examining whether pituitary down-regulation was achieved with E₂ < 25 pg/ml, luteinizing hormone (LH) < 5 U/L, the diameter of the ovarian follicles < 5mm, and an endometrial thickness < 5 mm. Patients who met these inclusion criteria commenced ovarian stimulation with gonadotropin of 100–300 IU daily until the day of hCG injection. The starting dose of gonadotropin was determined according to the body mass index (BMI), ovarian antral follicle count (AFC), ovarian reserve, and ovarian response in the past IVF/ICSI cycles. The dose could be adjusted for hormonal level changes and the size of the oocytes. Triggering of final follicular maturation was performed with 250µg recombinant hCG as a trigger when at least one follicle had a diameter of 18 mm or more or at least two follicles had a diameter of 17mm or more. After 35–38 hours of administration, follicles were aspirated under the guidance of transvaginal ultrasound. Embryo transfer was conducted on day five after oocyte retrieval. Clinical pregnancy was determined by visualizing a gestational sac on ultrasound at 6 weeks of gestation. Live birth was defined as having at least one alive baby born after 28 weeks of gestation.

Statistical analysis

R software (version 4.12), SPSS (version 25.0), Stata (version 15.0), GraphPad Prism (version 9.0) and MATLAB (version 2020a) were used for statistical analyses. Categorical variables were expressed as frequencies with percentages and continuous variables were shown using the medians [IQR]. Chi-square test and Fisher test were used to compare the categorical data. Mann-Whitney U test was used to compare continuous variables without normal distribution. A Scatterplot was used to study and visualize the associations between the 25(OH)D concentration, clinical pregnancy rate and birth rate. Collinearity diagnostics of variables were used to ensure the feasibility of binary logistic regression. Heatmaps were applied to visualize the associations of the combination of age and 25(OH)D concentration with CPR and LBR. The interaction analysis of binary logistic regression was used to demonstrate the absence of interactions between two variables. A binary logistic regression model was used to further explore the association between the 25(OH)D concentration, CPR and LBR adjusting other independent variables. Restricted cubic spline (RCS) was used to study the nonlinear relationship between the 25 (OH) D concentration and pregnancy outcomes. All statistical tests were bilateral, and statistically significant differences were inferred when P-values were less than 0.05.

The 612 patients were divided into two cohorts according to their serum 25(OH)D concentrations: ༜30.0 ng/ml (insufficient group) and > 30.0 ng/ml (sufficient group). The 91.53% of women (560) were in the deficient group, while 8.50% of women (52) were in the sufficient one. There was no significant difference in the distribution of age, BMI, infertility type, duration of infertility, and AFC in the two groups (P > 0.05 respectively), Table 1.

Table 1

Baseline characteristics of participants in deficient and sufficient cohorts
Variable	Total (%)	Deficient (%) [IQR]	Sufficient (%) [IQR]	P
Total	612	560	52
Age (Year)				0.276^a
20.00–30.00	261(42.65)	27.2(43.39)[3.3]	27.8(34.62)[3.67]
30.00–35.00	229(37.42)	32.1(37.14)[2.5]	31.8(40.38)[2.0]
≥ 35.00	122(19.93)	36.9(19.46)[2.9]	38.5(25.0)[1.9]
BMI (kg/m²)				0.581^a
༜18.50	66(10.78)	17.78(10.89)[0.84]	18.05(9.62)[0.17]
18.50–25.00	436(71.24)	21.48(71.43)[2.82]	21.16(69.23)[3.28]
25.00–30.00	98(16.01)	26.49(15.54)[1.85]	27.1(21.15)[1.61]
≥ 30.00	12(1.96)	31.64(2.14)[0.9]	None(0.0)[None]
Gravid (%)				0.491^b
Primiparity	221(36.11)	205(36.61)	16(30.77)
Multiparity	391(63.89)	355(63.39)	36(69.23)
Duration of infertility (Year)				0.709^a
1–4	453(74.02)	2.0(73.93)[2.0]	2.0(75.0)[2.0]
5–8	127(20.75)	6.0(21.07)[1.0]	7.0(17.31)[3.0]
9–12	28(4.58)	10.0(4.46)[1.0]	10.0(5.77)[1.5]
13–16	4(0.65)	13.0(0.54)[0.5]	16.0(1.92)[0.0]
AFC (number)				0.805^a
༜9	56(9.15)	6.0(8.75)[2.0]	5.0(13.46)[1.5]
9–19	350(57.19)	14.0(57.5)[5.0]	13.5(53.85)[5.0]
༞19	206(33.66)	24.0(33.75)[6.0]	25.0(32.69)[5.0]
AFC, antral follicle counting; BMI: body mass index; IQR = interquartile range; CPR: Clinical pregnancy rate
Unless otherwise stated, values are presented as median (interquartile)
^a Mann-Whitney U test.
^b Pearson χ²
*Indicates a significant (P < 0.05) difference between deficient and sufficient cohorts
**Indicates an extremely significant (P < 0. 001) difference between deficient and sufficient cohorts

The two groups were further divided into four subgroups using the following thresholds: ༜20.0 ng/ml (G1), 20.0–25.0 ng/ml (G2), 25.0–30.0 ng/ml (G3)], > 30.0 ng/ml (G4)]. Table 2 and Table 3 are presenting the baseline characteristics in pregnancy outcomes. The distribution of age was significantly different in pregnancy outcomes (P < 0.001 CPR and LBR) and in the infertility type in LBR (P < 0.004). Women of younger age or primary infertility may present better pregnancy outcomes compared to women with acquired infertility.

Table 2

Characteristics of cohorts’ outcome about CPR
Variable	Total (%)	CPS (%) [IQR]	CPF (%) [IQR]	P	CPR
Total	612	371	241		60.62
25(OH)D C(ng/ml)				0.592^a
0–20.00	284(46.40)	15.64(45.01) [3.46]	16.58(48.55) [3.76]		58.80
20.00–25.00	170(27.78)	22.30(27.49) [2.66]	22.12(28.22) [2.31]		60.00
25.00–30.00	106(17.32)	26.88(16.98) [2.08]	26.78(17.84) [2.14]		59.43
≥ 30.00	52(8.50)	32.54(10.51) [3.07]	35.08(5.39) [2.91]		75.00
Age (Year)				< 0.001^a**
20.00–30.00	261(42.65)	27.1(47.44) [3.40]	27.5(35.27)[3.0]		67.43
30.00–35.00	229(37.42)	31.9(37.74)[2.3]	32.1(36.93)[2.5]		61.14
≥ 35.00	122(19.93)	37.3(14.82)[3.4]	36.9(27.8)[2.6]		45.08
BMI (kg/m²)				0.514^a
༜18.50	66(10.78)	17.87(9.7)[0.48]	17.71(12.45)[0.86]		54.55
18.50–25.00	436(71.24)	21.41(73.85)[2.86]	21.48(67.22)[2.73]		62.84
25.00–30.00	98(16.01)	26.65(15.09) [1.67]	26.55(17.43) [1.79]		57.14
≥ 30.00	12(1.96)	31.18(1.35) [1.03]	31.64(2.9) [0.57]		41.67
Infertility type (%)				0.058^b
PI	221(36.11)	145(39.08)	76(31.54)		65.61
AI	391(63.89)	226(60.91)	165(68.46)		57.80
Duration of infertility (Year)				0.258^a
1–4	453(74.02)	2.0(77.36) [2.00]	2.0(68.88) [2.00]		63.58
5–8	127(20.75)	6.0(18.33) [2.00]	6.0(24.48) [1.00]		53.54
9–12	28(4.58)	10.0(3.77) [1.00]	10.0(5.81) [0.75]		50.00
13–16	4(0.65)	14.5(0.54) [1.50]	13.5(0.83) [0.50]		50.00
AFC (number)				0.045^a*
༜9	56.0(9.15)	6.5(9.16) [1.00]	6.0(9.13) [2.50]		60.71
9–19	350(57.19)	14.0(54.72) [5.00]	14.0(61.00) [5.50]		58.00
༞19	206(33.66)	24.5(36.12) [6.75]	23.0(29.88) [4.00]		65.05
CPS, Clinical pregnancy success; CPF, Clinical pregnancy failure; 25(OH)D C: 25(OH)D concentration; AFC, antral follicle counting; BMI: body mass index; PI, Primary infertility; AI, acquired infertility; IQR = interquartile range; CPR: Clinical pregnancy rate
Unless otherwise stated, values are presented as median (interquartile)
^a Mann-Whitney U test.
^b Pearson χ²
*Indicates a significant (P < 0.05) difference between CPS and CPF
**Indicates an extremely significant (P < 0. 001) difference between CPS and CPF

Table 3

Characteristics of cohorts’ outcome about BR
Variable	Total (%)	LB (%) [IQR]	ABN (%) [IQR]	P	BR
Total	612	341	271		55.72
25(OH)D C(ng/ml)				0.578^a
0–20.00	248(46.41)	15.63(44.87)[3.47]	16.52(48.34)[3.6]		53.87
20.00–25.00	170(27.78)	22.41(27.86)[2.7]	2.15(27.68)[2.2]		55.88
25.00–30.00	106(17.32)	26.96(17.01)[2.23]	26.58(17.71)[1.93]		54.72
≥ 30.00	52(8.5)	32.54(10.26)[3.47]	34.57(6.27)[3.06]		67.31
Age (Year)				< 0.001^a**
20.00–30.00	261(42.65)	27.0(49.56)[3.4]	27.55(33.95)[2.95]		64.75
30.00–35.00	229(37.42)	31.9(37.54)[2.45]	32.1(37.27)[2.3]		55.9
≥ 35.00	122(19.93)	37.45(12.9)[3.4]	36.8(28.78)[2.5]		36.07
BMI (kg/m²)				0.237^a
༜18.50	66(10.78)	17.83(9.97)[0.57]	17.71(11.81)[0.85]		51.52
18.50–25.00	436(71.24)	21.48(74.78)[2.85]	21.45(66.79)[2.7]		58.49
25.00–30.00	98(16.01)	26.64(14.37)[1.71]	26.62(18.08)[1.88]		50.0
≥ 30.00	12(1.96)	31.18(0.88)[0.53]	31.64(3.32)[0.78]		25.0
Infertility type (%)				0.004^b*
PI	221(36.11)	140(41.06)	81(29.89)		63.35
AI	391(63.89)	201(58.94)	190(70.11)		51.41
Duration of infertility (Year)				0.097^a
1–4	453(74.02)	2.0(78.01)[2.0]	2.0(69.0)[2.0]		58.72
5–8	127(20.75)	6.0(18.18)[2.0]	6.0(23.99)[1.0]		48.82
9–12	28(4.58)	10.0(3.23)[1.0]	10.0(6.27)[1.0]		39.29
13–16	4(0.65)	14.5(0.59)[1.5]	13.5(0.74)[0.5]		50.0
AFC (number)				0.066^a
༜9	56(9.15)	7.0(8.8)[1.75]	6.0(9.59)[2.0]		53.57
9–19	350(57.19)	14.0(55.13)[5.0]	14.0(59.78)[5.75]		53.71
༞19	206(33.66)	25.0(36.07)[6.0]	23.0(30.63)[4.5]		59.71
LB, Live birth; ABN, abortion; 25(OH)D C: 25(OH)D concentration; AFC, antral follicle counting; PI, Primary infertility; AI, acquired infertility; BMI: body mass index; IQR = interquartile range;
Unless otherwise stated, values are presented as median (interquartile)
^a Mann-Whitney U test.
^b Pearson χ²
*Indicates a significant (P < 0.05) difference between LB and ABN
**Indicates an extremely significant (P < 0. 001) difference between LB and ABN

Figure1. shown that CPR and LBR in G4 were significantly higher than those in G1-G3, suggesting that there might be a nonlinear relationship between the 25(OH)D concentration and both CPR and LBR.

As shown in Table 4, there was no multicollinearity among the respective variables (all VIF < 5) according to multivariable analysis.

Table 4

Multicollinearity test results of all variables used for logistic regression
Variable	Tolerance	VIF
25(OH)D C(ng/ml)	0.973	1.028
Age (Year)	0.850	1.176
BMI (kg/m²)	0.970	1.031
Infertility type (%)	0.913	1.096
Duration of infertility (Year)	0.940	1.063
AFC (number)	0.943	1.061
CPR: Clinical pregnancy rate

Heatmaps were provided to visualize the relationship between age, 25(OH)D concentration and CPR (Fig. 2A) and LBR (Fig. 2B). As shown in Fig. 2, younger participants or ones with higher 25(OH)D concentrations presented higher CPR or LBR. However, when binary logistic regression models were used and considering interaction terms to explore the interaction between the variables with significant differences in baseline characteristics (Table 1.) and 25(OH)D concentration, the results showed that neither age nor gravid had an interaction with 25(OH)D concentration. (P > 0.05 in Fig. 3.).

The results of the association between 25(OH)D concentration and CPR in our study are shown in Table 5. In the unadjusted model, P for Trend = 0.112, taking G4 as the reference, P < 0.05 for G1, and its Odds Ratios (95% CI) was 0.476 (0.243–0.930)., The results of binary logistic regression analysis showed statistically significant differences for the model adjusted for Age and AFC (Model1) and the model adjusted for all covariates (Model2). P-values for the trendlines in Model 1 and Model 2 were 0.038 and 0.043, respectively. The Odds Ratios (95% CI) of G1, G2 and G3 in Model1 against G4 were 0.417 (0.220–0.835), 0.472 (0.233–0.960) and 0.469 (0.223–0.990), respectively. In model 2, the Odds Ratios (95% CI) of G1, G2 and G3 against G4 were 0.414 (0.208–0.824), 0.457 (0.224–0.932) and 0.457 (0.215–0.971), respectively.

Table 5

**Logistic regression analyses of the relationship between concentration of 25(OH)D and CPR**
Variable	Crude			Model I^a
	OR (95%CI)	P-Value		OR (95%CI)	P-Value		OR (95%CI)	P-Value
Groups^&
G1	0.476(0.243–0.930)		0.030	0.417(0.220–0.835)		0.012	0.414(0.208–0.824)		0.012
G2	0.500(0.249–1.006)		0.052	0.472(0.233–0.960)		0.038	0.457(0.224–0.932)		0.031
G3	0.488(0.234–1.021)		0.057	0.469(0.223–0.990)		0.047	0.457(0.215–0.971)		0.042
G4	1.000			1.000			1.000
P for trend			0.112			0.038			0.043
The last group is used as the respective reference group
a: Adjust for age and AFC
b: Adjust for age, BMI, AFC, gestation and duration of infertility
OR, odds ratio; CI, confidence interval;
&: All groups are based on Table 1.

Table 6. shows the results of exploring the relationship between 25(OH)D concentration and Birth Rate in the participating population. In the unadjusted model, the relationship between each group and Birth Rate was not found to be statistically significant, with the p-value of the trendline being 0.179. The p-value of the trendline of the binary logistic regression analysis for adjusted for Age, AFC and gravid model 1 was 0.036. G4 as the reference, A statistically significant difference was found between G1 and G4, which was used as reference, regarding their association with LBR, with the odds ratio (95% CI) being 0.469 (0.246–0.895). The p-value of the trendline of Model 2, which was adjusted for all covariates, was 0.052. A statistically significant difference was found between G1 and G4, which was used as reference, regarding their association with LBR, with the odds ratio (95% CI) being0.472 (0.246–0.904).

Table 6

**Logistic regression analyses of the relationship between concentration of 25(OH)D and BR**
Variable	Crude			Model I^a
	OR (95%CI)	P-Value		OR (95%CI)	P-Value		OR (95%CI)	P-Value
Groups^&
G1	0.567(0.304–1.059)		0.075	0.469(0.246–0.895)		0.022	0.472(0.246–0.904)		0.023
G2	0.615(0.320–1.183)		0.145	0.565(0.288–1.106)		0.096	0.552(0.281–1.086)		0.085
G3	0.587(0.293–1.175)		0.132	0.567(0.278–1.155)		0.118	0.540(0.264–1.106)		0.092
G4	1.000			1.000			1.000
P for trend			0.179			0.036			0.052
The last group is used as the respective reference group
a: Adjust for age, AFC, infertility type
b: Adjust for age, BMI, AFC, gestation and duration of infertility
OR, odds ratio; CI, confidence interval;
&: All groups are based on Table 2.

Figure 4. depicts the dose-response curve when the predicting the Odds Ratio value of clinical pregnancy and live birth using the binary logistic regression model. Model 2 was used as the dependent variable and 25(OH)D concentration (continuous variable) of the participating population was used as the independent variable. The fitting method used was the restricted cubic spline curve (RCS). The dose-effect relationship curve suggested that there was a nonlinear positive correlation between the 25(OH)D concentration and pregnancy outcome (P for nonlinear < 0.001, respectively). CPR and LBR increased with the increase of 25(OH)D concentration. For example, taking sufficient 25(OH)D concentration (30ng/ml) as reference, the women with a sufficient 25(OH)D concentration (30ng/ml) presented 1.07 times increased clinical pregnancy rates and 1.05 times increased live birth rates compared to women with an insufficient 25(OH)D concentration (25ng/ml). (OR_25ng/ml,) [95%CI] = 0.935[0.932–0.938] and 0.947[0.945–0.950], P < 0.001, respectively). In the range of > 30 ng/ml, the positive correlation effect of 25(OH)D concentration on CPR and LBR was stronger than the one below 30 ng/ml. However, when the serum 25(OH)D concentration was lower than 25ng/ml, the OR value remained relatively stable. (OR_20ng/ml) [95%CI] = 0.931[0.926–0.936] and 0.941[0.936–0.946], P < 0.001, respectively), (OR_15ng/ml,) [95%CI] = 0.913[0.909–0.918] and 0.913[0.909–0.917], P < 0.001, respectively).

Infertility is a disease and social problem. According to the reports⁹, about 10–12% of couples are suffering from infertility worldwide. In China, relevant studies have shown^9–11 that the prevalence of infertility greatly varies among different regions, with the prevalence among women of childbearing age ranging from 6.7–25%. One of the current methods to solve this problem is to conduct IVF for women of childbearing age with confirmed infertility. Therefore, researchers have been exploring how to obtain a good pregnancy outcome of IVF/ICSI-ET conveniently and effectively.

Current systematic studies suggest that Vitamin D may have some positive effects during normal pregnancy. For example, Vitamin D can promote the development of the nervous^17–19, motor^8,17,20, and respiratory²¹ systems of the fetus, respectively, and regulate the immunity²² anti-infection, and allergy. In contrast, lower serum 25(OH)D in pregnant women has been linked to a higher risk of preterm birth (PTB) or intrauterine growth retardation (IUGR) ²³.

Our study set 30ng/ml as sufficient 25(OH)D concentrations, as defined by the Italian Association of Clinical Endocrinologists in 2018²⁴, which recommended maintaining the 25(OH)D levels above 30 ng/mL (75 nmol/L). Furthermore, Rudick B reported racial differences in the effect of 25(OH)D concentrations on clinical pregnancy rates. However, the 25(OH)D deficiency was associated with a lower pregnancy rate among non-Hispanic whites, but not among Asians, with the authors suggesting that this might be attributed to the lower success rate of IVF-ET in Asia²⁵. The present study is thus the first to investigate the relationship between serum 25(OH)D levels and CPR in IVF/ICSI-ET in Chinese populations. 25(OH)D was significantly positively associated with CPR. This result has been supported by similar studies. For instance, a meta-analysis showed²⁶ that among all women who underwent IVF/ICSI-ET in their analysis, women with ample 25(OH)D were more likely to have a good clinical pregnancy outcome than women with deficient or insufficient 25(OH)D. Furthermore, Jing Zhao et al. reached a similar conclusion that 25(OH)D sufficiency increased live birth rates as an IVF/ICSI-ET outcome²⁷. However, to the best of our knowledge, none of the existing studies have reported the nonlinear positive correlation yet.

The results of the present study were supported by many molecular mechanisms. Vitamin D promotes placental calcium transport, stimulating the expression of prolactin and decidualization, regulating the expression of HOXA 10, a target gene that has been associated with the implantation process^28–32. Another experimental study reported that the lack of the VDR gene is associated with gonadal insufficiency, down-regulation of aromatase gene expression, low aromatase activity, hypergonadotropic hypogonadism^32,33, and features of estrogen deficiency, such as bone malformations, uterine hypoplasia, impaired folliculogenesis, and infertility^32,34.

Furthermore, our study showed that age remains an important factor in IVF/ICSI-ET. In the heat map of Fig. 2A the OR was found to exceed 10 and the color changed 30 times faster for women younger. Changes in the same 25(OH)D levels had a more positive effect on changes in pregnancy outcomes in women under 30 years of old. A similar conclusion related to LBR is shown in Fig. 2B. Therefore, we hypothesize that women below the age of 30 are more sensitive to Vitamin D deficiency.

However, our research results were still controversial. Jason M. Franasiak's study showed that the effect of 25(OH)D on pregnancy outcomes was insignificant³⁵. Abbas Aflatoonian suggested that 25(OH)D deficiency treatment did not relate to a higher pregnancy rate in the cycle of frozen-thawed embryo transfer³⁶. Arne van de Vijver³⁷ and Liu Jiang¹³ also reached the similar conclusion of no significant correlation between the two. The process of IVF/ICSI-ET is complex, and many factors can affect outcomes. Combined with our findings, it is suggested that this difference could be explained from the perspective of age, since the clinical pregnancy outcomes of women over 30 years old, especially the elderly pregnant women (age ≥ 35 years old), are affected by other more important factors implying the presence of a bias in our model. For example, research by Michaël De Brucker has shown that clinical pregnancy rates in older pregnant women were driven by several complications. The incidence of pregnancy-induced hypertension was only 6–8% in the general pregnant women group, while it increased to 15% in the elderly pregnant women³⁸. In addition, it is obvious that women’s fertility decreases with the increase in age³⁹ with a notable change being the deterioration of the ovarian reserve function with age. Therefore, AFC, as one of the indicators for evaluating the ovarian reserve function⁴⁰, showed a downward trend with aging⁴¹. Moreover, the decline in ovarian function adversely affects the outcome of IVF/ICSI-ET. Therefore, if the patients were not grouped by age, it would have been very likely to obtain different results. Moreover, as shown in our study, the relationship between 25(OH)D and pregnancy outcomes of IVF/ICSI-ET patients is nonlinear. This means that if the patient’s 25(OH)D levels were not well grouped or mainly distributed below 25ng/ml, it would be difficult to observe the correct conclusion.

In summary, our study was the first case study on the Chinese population to propose the CPR and LBR as the research outcome of IVF/ICSI-ET, that explored the trends of pregnancy outcomes under the combined effects of covariates such as age. According to PubMed, we were the first to describe this nonlinear relationship using RCS curves. This was important for the subsequent development of IVF/ICSI-ET and for patients to obtain a good pregnancy outcome. To some extent, it bridged and explained previous controversial views in this field. However, our study still possesses some limitations. First, it only included female patients administered by the Second Affiliated Hospital of Wenzhou Medical University. Second, it had been suggested⁴² that seasonal variations may affect circulating 25-(OH)D levels, and we did not rule out these possible interferences in this study. In addition, there were many factors affecting the pregnancy outcome. For example, measured in early pregnancy 25(OH)D levels are likely to fail to better represent 25(OH)D levels in late pregnancy, weakening thus the link between 25(OH)D levels and LBR compared to CPR. Further research could promote understanding of the role and influence of 25-(OH)D in IVF/ICSI-ET better and finally contribute to the identification of methods to improve IVF/ICSI-ET pregnancy outcome.

Serum 25(OH)D level was significantly related to pregnancy outcome(CPR&LBR)in Chinese women receiving IVF/ICSI-ET with a nonlinear positive correlation. When 25(OH)D levels exceeded 25 ng/ml, the correlation appeared stronger, while insufficient 25(OH)D (under 30ng/ml) deliver a worse pregnancy outcome. However, their does not exist substantial evidence to allow considering different 25(OH)D levels in distinct ages.

IVF-ET/ICSI: In Vitro Fertilization - Embryo Transfer/Intracytoplasmic Sperm Injection

IVF-ET: In Vitro Fertilization - Embryo Transfer

ICSI: Intracytoplasmic Sperm Injection

CPR: Clinical Pregnancy Rate

LBR: Live Birth Rate

RCS: Restricted cubic spline

OR: Odds ratio

BMI: Body mass index

CNHS: Chinese national nutrition surveillance

IGF-BP-1: Insulin-like growth factor binding protein 1

LC-MS: Liquid chromatography-mass spectrometry

GnRH-a: Gonadotropin-releasing hormone agonist

hCG: Human chorionic gonadotrophin

CO₂: Carbon dioxide

O₂: Oxygen

ICM: Inner cell mass

IQR: Inter-quartile range

CIs: Confidence intervals

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

The authors would like to express their gratitude to EditSprings (https://www.editsprings.cn ) for the expert linguistic services provided.

Funding

This study was supported by Clinical Research Foundation of the 2nd Affiliated Hospital of Wenzhou Medical University (SAHoWMU-CR2017-07-124).

Author information

Affiliations

Center for Reproductive Medicine and Infertility, The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang, China

Haihong Chi, Junzhao Zhao.

Second School of Clinical Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, China

Zhengchen Yu & Jinrui Xie & Zhenzhen Lu& Yiqun Sun& Luxuan Xu& Yuxi Zhou

School of Dentistry, Wenzhou Medical University, Wenzhou, Zhejiang, China

Peng Wang

First School of Clinical Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, China

Yinkai Hu

Contributions

Z.Y., P.W. and J.Z. contributed to the concept and design of the study. Z.Y. conceived, analyzed results, and wrote/edited articles. P.W. and Y.H. tested and analyzed the results. Y.S. collected the data. J.X. and Y.S. provided assistance with certain aspects of data analysis. Z.L. and L.X. involved in the revision of the manuscript. H.C. and Y.Z. conceived the study and participated in its design, coordination, and article modification. Z.L. contributed to the revision of the manuscript. The author read and approved the final draft .

Corresponding authors

Correspondence to Haihong Chi, Junzhao Zhao

Ethics declarations

Ethics approval and consent to participate

This study complied with the Declaration of Helsinki and was approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University (LCKY2018-38).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Dataset Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Vitamin D level is associated with increased clinical pregnancy rate and live birth rate in Chinese women undergoing IVF/ICSI-ET: a retrospective cohort study

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Abstract

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Introduction

Method

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Serum 25(OH)D measurement

IVF/ICSI-ET procedure

Statistical analysis

Result

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