Lean non-PCOS women with insulin resistance have adverse IVF outcomes with the decreased percentage of mature oocytes and blastocyst formation rate and increased peripheral blood B cell levels.

Insulin resistance (IR) is typically defined as decreased sensitivity or responsiveness to the metabolic action of insulin (1). IR is caused by a primary defect in insulin receptor signaling and a reduced insulin clearance rate, resulting from decreased hepatic insulin extraction (2). It is commonly associated with obesity, hypertension, cardiovascular disease, and typically type 2 diabetes. It is reported that about 50% to 70% of women with PCOS have IR (3). Infertile women with PCOS had higher levels of fasting insulin, and the resultant hyperinsulinemia plays a role in the pathogenesis of reproductive disorders (4–6).

Hyperinsulinemia disrupts the intrafollicular microenvironment during folliculogenesis and reduces the rate of fertilization and embryonic development potential during the natural and ovarian stimulation cycles (7, 8). Insulin signaling in the uterus controls gene expression and glucose utilization and affects the decidualization process to facilitate implantation (9). IR and free androgen index correlate with total ovarian follicle count in non-PCOS women who underwent IVF-ET treatment. It is suggested that the higher level of IR and androgen can positively affect short-term follicle development and benefit responses to exogenous gonadotrophin stimulation while increasing the risk of OHSS in non-PCOS women (10).

There is increasing evidence at the cellular level showing that inflammation is a critical factor for obesity-induced IR. The tissue-resident immune cells, especially adipose-tissue resident cells, play a major role in regulating obesity-induced inflammation. On the other hand, cellular and molecular factors in adipose tissue regulate obesity-induced inflammation and IR (11). Elevated inflammatory cytokines, including interleukin (IL)-17 and IL-6, could cause a subclinical inflammatory state for a prolonged time. Abnormal inflammatory responses decrease glycolipid metabolism, increase IR, and affect ovulation and fertilization, resulting in polycystic ovary syndrome (PCOS) characterized by oligomenorrhea and irregular ovulation (12).

Obesity is a global epidemic related to numerous health concerns, including reproductive disorders. Central obesity is considered an independent risk factor for early miscarriage (13). Obese individuals exhibit increased estrogen concentrations due to aromatase overexpression in the adipose tissue. Consequently, higher estrogen level causes anovulation via the hypothalamus-pituitary-ovary axis (14). On the other hand, weight loss through lifestyle changes or bariatric surgery positively affects hormonal parameters and ovulation rates (15, 16).

Most of the studies found the effect of IR on IVF outcome in women with overweight/obese PCOS, using homeostasis model assessment for insulin resistance (HOMA-IR) as the index to assess IR. The HOMA index was calculated as HOMA = (fasting insulin uIU/ml * fasting glucose mmol/l)/22.5.HOMA-IR has been reported to effectively predict IR in the overweight-obese PCOS population with a cutoff of 2.62 or more (AUC 84.1%) (17). However, HOMA-IR was found not to be reliable or predictable in detecting IR in lean women, who had neither fasting hyperinsulinemia nor increased basal hepatic glucose production (18). Recently, the HOMA-M₁₂₀ was reported as a reliable and straightforward measure of IR for lean European and Asian women with PCOS (17, 19). HOMA-M₁₂₀ was calculated as (post-load 2-hour insulin uIU/ml * post-load 2-hour glucose mmol/l)/22.5. IR was diagnosed when HOMA-M₁₂₀>12.8. Lean women with HOMA-M₁₂₀>12.8 were considered as IR group, and the others were non-IR group (17). Previous studies have focused on the adverse effect of IR on IVF outcome in women with overweight/obese PCOS. IR’s impact on reproductive outcome in lean PCOS has also been reported (20). Therefore, doctors pay more attention to the diagnosis and treatment of IR in obese and non-obese PCOS patients, metformin for example,to improve IVF outcome.It is noteworthy that IR can also occur in infertile women who had a regular menstrual cycle without polycystic ovaries, which has been reported before (10). But there are no researches about whether IR in lean patients without PCOS adversely affects IVF outcomes. Hence, in this study, we determined to investigate IR by HOMA-M₁₂₀ in lean women without PCOS and analyze the IR effect on IVF outcomes and related cellular and endocrine factors.

In this study, we found that IR is associated with a slower response to ovulation induction, poor oocyte maturation, and decreased proportion of freezable embryos in lean and non-PCOS women who underwent IVF treatment, with increased B cell levels as compared to non-IR women.

In this study, the prevalence of subclinical hypothyroidism was significantly higher in the IR group than the non-IR group among lean women without PCOS, which is consistent with the previous study demonstrating that the HOMA-M₁₂₀ index was significantly increased in women with subclinical hypothyroidism (23). There are several possible mechanisms to explain the observed relation between low-normal thyroid function and IR. Insulin might influence thyrotropin-releasing hormone (TRH) and TSH when modulating glycemic status (24), and subclinical hypothyroidism is associated with decreased insulin sensitivity and glucose tolerance, partially due to a decreased insulin ability to increase glucose utilization mainly in the muscle (25).

We have demonstrated that the retrieved number of oocytes was higher in the IR group than the non-IR group, although the difference did not reach a significant level, which is consistent with the previous study (10). Insulin promotes primordial to primary follicle transition (26). Additionally, the response of granulosa cells to FSH during the gonadotropin-dependent stage of folliculogenesis can be enhanced by insulin growth factors (27). Therefore, in the IVF setting, the risk of developing multifolliculogenesis or ovarian hyperstimulation syndrome to exogenous gonadotropin stimulation is higher in IR than non-IR patients (28).

In our study, the percentage of mature oocytes per oocytes retrieved and the percentage of freezable blastocysts per residual embryos were significantly lower than those of women in the non-IR group. Previous study found insulin could stimulate theca cell androgen production, elevating serum free testosterone levels, so hyperinsulinemia will increase the local production of androgens (29). It is proved that high level of androgen can interfere with fertilization and cleavage rates of in vitro–matured oocytes, which decrease the number of mature oocytes and blastocyst formation rate (30). In our study, the percentage of mature oocytes per oocytes retrieved and the percentage of freezable blastocysts per residual embryos were significantly lower than those of women in the non-IR group. This is consistence with previous study. But the direct mechanisms of hyperinsulinemia adverse mature oocytes and blastocyst need further study.

As metformin often induces side effects, new integrative strategies have been proposed to treat insulin resistance, such as the use of inositols. Myo-inositol (MYO) and d-chiro-inositol (DCI) are two inositol stereoisomers in humans. MYO is the precursor of inositol triphosphate, a second messenger that regulates thyroid-stimulating hormone (TSH) and FSH as well as insulin. Several preliminary studies suggest that a deficiency of D-chiro-inositol (DCI) containing IPG might be at the basis of insulin resistance (31). In fact Genazzani et al. reported that MYO administration can not only decrease fasting insulin plasma levels in obese patients (32) but also improve insulin sensitivity in non-obese PCOS patients (33).

Finally, in contrast to the non-IR group, the IR group had a significantly higher level of peripheral blood B lymphocytes. Since there is no difference in autoimmunity between the IR and non-IR groups, increased B cells in the IR group may reflect the expansion of the functional subset of B cells. Previous study found B cells can worsen glucose tolerance by production of IgG antibodies and activation of proinflammatory macrophages and T cells in mice model. Depletion of B cells in mice can ameliorate glucose tolerance and fasting insulin. However, the return of B cells will exert their detrimental effects on glucose tolerance again. These results indicates B cells play a role in insulin resistance (34). In our study, we found B cells were increased in IR group, this was consistence with previous studies. Further studies are required to confirm the mechanism of the speculation.

Previously, we reported that women with low vitamin D levels had higher peripheral blood B-cell proportion and T helper/T cytotoxic cell ratios than those of normal vitamin D (35). In this study, vitamin D levels were not different between the two study groups. Maybe the IR in lean women with non-PCOS didn’t have vitamin D deficiency tendency.

The study is limited since the underlying mechanism of higher B lymphocyte count in the peripheral blood of women with IR was not explored further and auto-antibodies to insulin and its receptors were not investigated. Since the genetic study of the embryos were not made in all cases, IR and genetic abnormalities of the embryos were not thoroughly investigated.

Currently, the independent role of IR in IVF outcomes is not defined well (30, 36). Our study demonstrated the association between IR and adverse IVF outcomes in the lean non-PCO population. Possible use of anti-glycemic agent during the IVF cycle should be investigated in the future in women with IR without obesity or PCOS.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethical Committee of the First Affiliated Hospital of USTC. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

All authors qualify for authorship by contributing substantially to this article. HW and LW were responsible for study design and conception, data analysis, the first draft of the article, review, and approval of revisions, and the final article. JK-K interpreted the data and revised the manuscript. XF and YZ were responsible for recruitment, data collection, and article review. All authors contributed to the article and approved the submitted version.

This study was supported by the National Natural Science Foundation (No. 81601353, No.82071650).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.