Subjects and procedures
150 patients were enrolled into this study. All patients provided a signed in-formed consent. The mean age of the 150 patients was 30.02 years with a standard deviation of 4.58 years. The Body mass index (BMI) is 18.4-31.6 (22.47±3.15) Kg/m2. The duration of infertility is 1-10 (3.62±2.13) years.
(1)Inclusion criteria: Patients in childbearing age; patients who firstly underwent GnRH-a prolonged protocol IVF-ET due to tubal factors from January 2018 to December 2018.
(2)Exclusion criteria: Patients with complaints of discharge, itching, burning and dysuria; patients who undergo vaginal douching, or have sexual intercourse within 72 h before specimen acquisition; patients who received systemic antibiotics within 4 weeks; patients who received medication of sexual hormones within 3 months; patients with physical diseases, including cardiovascular, respiratory, endocrine, chronic autoimmune or blood system diseases, etc.
Ovarian stimulation protocol and IVF-ET
All patients in this study was performed GnRH-a prolonged protocol IVF-ET. It is composed of ovarian suppression with a GnRH-a, followed by COH with recombinant and urinary-derived gonadotropins, followed by HCG administration to induce oocyte maturation. Pituitary down-regulation was achieved with a single full-dose injection of 3.75mg GnRH-a (Leuprolide Acetate; Shanghai Livzon Pharmaceutical co., LTD) on day 2 of the menstrual cycle. Successful pituitary down-regulation was confirmed with follicle diameter<8mm, serum estradiol (E2) <50pg/mL, serum LH<5IU/L, and endometrium thickness<5mm.
Then, COH with recombinant human follicle stimulating hormone (rFSH) ranging from 75 to 300 IU per day would start 32-38 days later. The dosage was determined according to patients’ BMI, antral follicular count (AFC) and basal follicle-stimulating hormone (FSH) level. When two leading follicles reached a mean diameter of 18 mm, 6000-8000 IU HCG (Livzon Pharmaceutical Group Inc., China) was used for a trigger.
Transvaginal oocyte retrieval was performed 36-37h after HCG administration. Fertilization was achieved using standard IVF or ICSI. Gametes and embryos were handled separately according to standard laboratory procedures. Embryo quality was analyzed according to the Istanbul consensus workshop on embryo assessment. Fresh embryo transfer was performed on day 3 or day 5 after fertilization. Embryo transfer was determined according to the embryo quality, the thickness and state of endometrium. All embryos were at least good quality (Grade B) with 7-9 cells and less than 10% fragmentation and even symmetry, or high-quality blastocyst.
Luteal phase support was sustained from the oocyte retrieval day and was continued until the day of serum HCG testing. For women with positive HCG data, luteal phase support was continued until 10 weeks of gestation.
Serum E2 concentration measurements
Blood was drawn, and serum prepared at three time points during each treatment cycle: the baseline; the first day of Gn; the trigger day.
Definition of clinical outcomes
The primary outcome was the live birth rate after fresh embryo transfer. The secondary outcomes include the clinical pregnancy rate, the implantation rate, the biochemical pregnancy rate, the early miscarriage rate.
Biochemical pregnancy was defined with the result of serum β-HCG≥10mIU/ml measured 11 Day after embryo transfer. Clinical pregnancy was defined as detection of gestational sac by transvaginal ultrasound scan 35 days after embryo transfer. The ongoing pregnancy was defined as detection of a viable fetus with fetal heartbeat at 11-12th week of gestation. The early miscarriage was defined as spontaneous abortions within 12 weeks of pregnancy. The live birth rate was classified as delivery of any viable infant after 24 weeks.
Live birth rate (LBR) per fresh transplantation cycle = the number of deliveries/the number of fresh embryo transfer cycles × 100%
Clinical pregnancy rate (CPR) = the number of clinical pregnancy patients/ovulation cycle patients × 100%
Implantation rate (IR) = the number of implantation embryos/the number of transferred embryos × 100%
Biochemical pregnancy rate (BPR) = the number of biochemical pregnancy patients/ the number of fresh embryo transfer patients × 100%
Early miscarriage rate (EMR) = the number of early miscarriage patients/the number of clinical pregnancy patients × 100%
Specimen acquisition and laboratory tests
Vaginal secretion collection
The secretion on the posterior fornix and upper 1/3 segment of the vagina was collected by rotating two sterile long cotton swabs to evaluate the vaginal micro-ecology. The swab 1 was taken on the day of pituitary down-regulation and the swab 2 was taken on the day of HCG administration, respectively. It also required menstruation to be clean for at least 3 days. Patients with LRTI were treated accordingly.
Evaluation of vaginal micro-ecology
Vaginal micro-ecology evaluation system (VMES) includes microscopic detection of flora density, flora diversity, dominant bacterial flora, pathogen, aerobic vaginitis (AV) score for AV, Nugent score for BV and the functional indicators.
(1) Low power microscope was used to observe the presence or absence of trichomonas in wet physiological saline, and AV score was performed at the same time.
(2) After Gram staining of secretion smear, 10×100 times oil microscope was used to check the flora density, diversity and predominant flora, and whether there were budding spores or pseudo hyphae, and Nugent score was carried out.
(A) Flora density: results were recorded as grades I-IV, according to the average number of bacteria in each visual field.
I:average number of bacteria is 1-9/field;
II:average number of bacteria is 10 to 99/field;
III:average number of bacteria is more than 100/field;
IV:bacteria aggregate into clusters or densely cover mucosal epithelial cells.
(B) Flora diversity: results were classified into grades I-IV, according to the species number of visible bacteria.
I:1-3 species of visible bacteria;
II:4-6 species of visible bacteria;
III:7-9 species of visible bacteria;
IV:more than 10 species of visible bacteria.
(C) Predominant flora: the largest number of the microorganism species.
(D) Clue cells, budding spores or pseudo hyphae were detected under a microscope by H&E staining.
(3) The five functional indicators: hydrogen peroxide (H2O2), sialidase, leukocyte esterase, beta-glucuronidase and coagulase were detected using Aerobic Vaginitis and Bacterial Vaginosis Diagnostic Strip Sets (Beijing Zhong Sheng Jin Yu Diagnostic Technology Co. Ltd).
Diagnostic criteria
- Flora density: “II” and “III” were defined as normal, while “I” and “IV” were defined as abnormal.
- Flora diversity: “II” and “III” were defined as normal, while “I” and “IV” were defined as abnormal.
- Predominant flora: when the dominant bacterial flora was Gram-positive rods, the predominant flora was normal; and other cases were defined as abnormal.
- Functional indicators: When H2O2 was positive, it was determined as normal function of Lactobacillus; when the other four items were negative, they were defined as normal.
- AV: A composite AV score of < 3 correspond to “no signs of AV”, 3-4 to “light AV”, 5-6 to “moderate AV”, and any score > 6 to “severe AV” (Table 1).
Table 1. Criteria for the microscopic diagnosis of AV [7]
(×400 magnification, phase contrast microscope)
AV score
|
LBG
|
No. of leukocytes
|
Proportion of toxic leukocytes
|
Background flora
|
Proportion of PBC
|
0
|
I and IIa
|
≤10/hpf
|
None or sporadic
|
Unremarkable or cytolysis
|
None or <1%
|
1
|
IIb
|
>10/hpf and ≤10/epithelial cell
|
≤50% of leukocytes
|
Small coliform bacilli
|
≤10%
|
2
|
III
|
>10/epithelial cell
|
>50% of leukocytes
|
Cocci or chains
|
>10%
|
Lactobacillary grades (LBG) (I) numerous pleiomorph lactobacilli, no other bacteria; (IIa) mixed flora, but predominantly lactobacilli; (IIb) mixed flora, but proportion of lactobacilli severely decreased due to increased number of other bacteria: (III) lactobacilli severely depressed or absent because of overgrowth of other bacteria. hpf: high power field. PBC: parabasal epitheliocytes.
- BV: The diagnostic criteria for BV are as follows: (a) total score 7-10, BV; (b) score 4-6, intermediate BV; sialidase positive as an auxiliary diagnostic indicator (Table 2).
Table 2.The Nugent scoring criteria[8]
Score
|
Lactobacillus morphotypes
|
Gardnerella and Bacteroides spp. morphotypes
|
Curved gram-variable rods
|
0
|
4+(>30)
|
0(0)
|
|
1
|
3+(5~30)
|
1+(<1)
|
1+(<1)or 2+(1~4)
|
2
|
2+(1~4)
|
2+(1~4)
|
3+(5~30)or 4+(>30)
|
3
|
1+(<1)
|
3+(5~30)
|
|
4
|
0(0)
|
4+(>30)
|
|
0, No morphotypes present; 1, <1 morphotype present; 2, 1 to 4 morphotypes present; 3, 5 to 30 morphotypes present; 4, 30 or more morphotypes present.
- Vulvovaginal candidiasis (VVC): microscopic examination of budding yeast,or hyphal forms.
- Trichomonas vaginitis (TV): microscopic examination of active trichomonas with numerous white blood cells.
- Normal vaginal micro-ecology (NVM): vaginal flora density grade II-III, flora diversity grade II-III, dominant bacterial flora is lactobacillus, Nugent and AV score ≤3, and absence of pathogens and negative specific enzymes.
- Abnormal vaginal micro-ecology (AVM): any one of vaginal microflora density, diversity, dominant bacteria, inflammatory reaction and vaginal microbial functional indicators is abnormal.
Table 3.Vaginal micro-ecology evaluation system [9]
Items
|
Normal
|
Abnormal
|
Morphological indicators
|
|
|
Flora density
|
grades II/III
|
grades I/IV
|
Flora diversity
|
grades II/III
|
grades I/IV
|
Predominant flora*
|
Large Gram-positive rods
|
Gram-positive cocci
Large Gram-negative rods
Small Gram-negative rods
|
Nugent score
|
1-3
|
≥4
|
AV score
|
<3
|
≥3
|
Pathogen
|
Negative
|
Fungus(budding yeast,or hyphal forms)and/or trichomonas
|
Vaginal pH
|
≥3.8 and <4.5
|
<3.8 or ≥4.5
|
Functional indicators
|
|
|
H2O2
|
Positive
|
Negative
|
Enzymes
|
Negative
|
Positive
|
*Large Gram-positive rods (Lactobacillus); Gram-positive cocci (Staphylococcus aureus, Staphylococcus epidermidis); Gram-negative rods (Escherichia coli).
Statistical analysis
Data were analyzed using IBM SPSS 25.0 statistical software. Quantitative data were described as mean ± standard deviation (Mean ± SD) or median. Results were analyzed using t tests for comparison between the study groups and the control group. Pearson χ2 or Fisher exact test were used for the comparison of proportion, as appropriate. Then, multivariate logistic regression was performed. P <0.05 was considered to indicate statistical significance.
Ethics statement
This study was a retrospective analysis of clinical outcomes, and the Institutional Review Board of the Affiliated Hospital of Qingdao University approved our analysis of the data.