Title: The Relationship between Genetic Variants Associated with Primary Ovarian
Insufficiency and Lipid Profile in Women Recruited from MASHAD Cohort Study
Version: 1 Date: 05 May 2021
Reviewer's report:
Thank you for the opportunity to review this interesting article examining the genetic variants
associated with premature menopause (PM) and lipid profiles. This study found that some PM
related SNPs were associated with dyslipidaemia. However, I have several major concerns about
the findings: (1) lack of details on the selection of 8 SNPs in this study – which genes and
any potential links with lipids?; (2) lack of details on the genotype distribution of these SNPs
between the two groups and whether they meet the condition of Hardy–Weinberg equilibrium;
(3) the association between SNPs and lipids did not show a consistent pattern of results – more
like random findings in the PM group and non-PM group based on P<0.05; (4) the results did
not adjust for important confounders BMI and smoking that strongly associated with age at
menopause and lipids, and the statistical power seemed low as the result of very wide confidence
intervals. The authors
concluded that it increased by five times, based on the CI of 1.32 to 20.94.
Specific comments in each section:
Introduction:
1. When we talk about premature menopause or primary ovarian insufficiency (POI), mostly
referring to spontaneous POI, which affects 1-3% of women. Surgical menopause due to bilateral
oophorectomy before age 40 is different, so please do not mix up these two types and age of
menopause together.
2. The authors stated that some genetic variants (MCM8, PRRC2A, and TMEM) are associated
with age at menopause, but weak study rationale to study these genes variants with lipid profiles.
We know there is an association between early menopause and cardiometabolic disease risk, but
what is the potential link of these genes with lipid profiles? If lack of a link, it raises a concern
of multiple simultaneous testing.
3. The section of 8 SNPs included in this study is not clear. What are the genes of these SNPs?
Whether they are also associated with estrogen regulation or lipid metabolisms.
Methods:
4. Please clarify the definition of healthy control. Were they just the women who did not have
premature menopause, or they were non-PM and free of certain conditions? Any criteria to match
the controls? Age-matched?
5. Please further clarify the exclusion criteria of history of genetic abnormalities and syndromes
associated with an early menopause is a part of their manifestation.
6. Please add details on the list of included PM-related SNPs.
7. Please add a table showing the genotype distribution of all the SNPs between the PM and non-
PM group, and test Hardy–Weinberg equilibrium for genotype frequencies in the non-PM group.
8. The multivariable model was only adjusted for age and physical activities. Although BMI
and smoking status were not significantly different between the PM and non-PM group, they are
important confounders for age at menopause and lipids. Failure to adjust for BMI and smoking
will not provide the actual relationship.
9. Due to the small sample size, the estimates had very wide confidence intervals. Please provide
the details of the power calculation to support the robustness of the findings.
Results:
10. One of the exclusion criteria was women over 40 years old, but the result showed that
participants had a mean age of 55 years. This did not make sense. Table 1 should add the mean
age (SD) separately for the PM and non-PM group.
11. The associations with lipids were not consistent – more like random findings based on p-
values <0.05 and very wide confidence intervals.
Minor edits:
12. The MASHAD study is a cohort study from 2010-2020 that were included  9704 participants
aged 35-65 years who will be follow-up exams every three years until 2020 – please use past
tense in the methods.
13. Inconsistent information on fasting time:
1) Blood samples were collected into vacutainer® plain tubes, and were taken after 14 hours fast.
2) Total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C)
and low-density lipoprotein cholesterol (LDL-C) levels were measured from serum which was
taken from participants 12 hours fasting using standard method.
14. Please remove: If HDL cholesterol levels <40 mg/dl (<1.04 mmol/l) for men, as all
participants were all women.