Evaluation of primary infertility and factors affecting repeated failures in assisted reproductive technology outcomes

Infertility profile of couple (2019–2022)

The male partner, a 36-year-old Marine Engineer, and the female partner, a 32-year-old software engineer, both have normal body mass index and no chronic health issues or substance abuse habits. His marine engineering job requires him to be away for 3–4 months several times a year. They reported no family history of fertility problems and genetic anomalies in their 1^st degree and 2^nd degree relatives. In 2019, semen analysis showed the sperm count was 33 million/mL, with 60% motility, but only 2% had normal morphology, leading to a diagnosis of teratozoospermia. The female partner had regular menstrual cycles and had not reported prior pregnancies, miscarriages or other reproductive issues. She was clinically diagnosed with primary infertility, while her 2019 tubal patency test confirmed patent fallopian tubes bilaterally, indicating no blockages.

History of unsuccessful IUI and IVF outcomes (2019–2022)

Before approaching the tertiary-level (Level 3) fertility clinic, the couple had undergone multiple ART cycles, including two intrauterine inseminations (IUI) and one conventional IVF cycle between 2019 and 2022 at two private fertility clinics (Level 2 fertility clinics). The couple faced repeated ART failures from 2019 to 2022. During the 2019–2022 period, the woman’s prolactin (PRL) levels had fluctuated between 3.7 and 42.28 ng/mL, with a normal level of 3.70 ng/mL on December 31, 2022. Hyperprolactinemia had likely impaired ovulation and follicular development.^[3] Elevated FSH levels (11.7 IU/L and 15.9 IU/L) indicated diminished ovarian reserve and might have disrupted ovulation through hypothalamic-pituitary-ovarian axis interference.^[2] The thyroid-stimulating hormone (TSH) level of 3.65 μIU/mL suggested mild hypothyroidism, which could have caused menstrual irregularities and infertility. The woman’s testosterone was low (0.48 nmol/mL), and estradiol (E2) was 50.8 pg/mL. Anti-Müllerian hormone (AMH) was 1.22 ng/mL, reflecting moderate ovarian reserve for her age (32 years). These hormonal imbalances due to the low AMH, high FSH, low testosterone, and hyperprolactinemia in the early follicular phase had likely contributed to suboptimal ovarian function, disrupted ovulation and poor ART outcome.^[2-4]

Diagnostic workup and history of ART cycles in the tertiary level fertility clinic (February 2023–September 2023)

In February 2023, the couple visited the tertiary-level fertility clinic to undergo advanced ART fertility treatment. The female partner presented with diminished ovarian reserve, while the male partner was diagnosed with teratozoospermia. ICSI was recommended for ART. The initial IVF cycle commenced with recombinant-FSH (rFSH) 150 IU and HMG 150 IU, but no follicular response was observed after 4 days of stimulation. The dosage was increased to 300 IU of HMG for 2 more days, but still, no follicle >10 mm or significant estrogen rise (65.83 pg/mL) was detected. Consequently, the cycle was cancelled.

Genetic analysis of three key reproductive genes, namely, LH/choriogonadotropin receptor (LHCGR), follicle-stimulating hormone receptor (FSHR), and estrogen receptor (ESR1), was undertaken to investigate potential causes of the poor ovarian response. The woman’s hormone profile revealed erratic fluctuations in LH and FSH levels, both critical for ovarian function.^[6,7] LHCGR mutations are known to cause anovulation and polycystic ovary syndrome, while FSHR mutations impair follicular development and are linked to conditions such as primary amenorrhea and IVF outcomes.^[8,9]

Genetic testing revealed the female partner’s homozygous wild-type LHCGR genotype at Exon 1 position (c.54_55ins6bp, rs4539842), which is associated with poor ovarian response, embryo implantation failures and increased IVF failure rates. FSHR analysis showed a homozygous wild-type G/G genotype at the 5’UTR (rs1394205), indicating increased androgen levels and diminished ovarian reserve. The Exon 10 polymorphisms (rs6165 and rs6166) suggested a need for higher doses of rFSH. Finally, ESR1 genotypes (rs9340799 and rs2234693) demonstrated a favorable ovarian response to controlled ovarian hyperstimulation (COH), suggesting better embryo quality and a higher probability of IVF success.^[8,9]

Integrating hormone profile and genetic polymorphism results in IVF-ICSI cycles at the tertiary-level fertility clinic

We integrated the genetic polymorphism analysis results of the woman into the optimization of her ART protocols for the IVF-ICSI cycle #1 (June, 2023). Variants of the LHCGR, FSHR, and ESR1 genes were used to tailor ovarian stimulation regimens and timing of embryo transfer for a personalized ART protocol.^[8,9] The deleterious effects of LHCGR gene variants were addressed with customized treatment, while FSHR variants indicated poor ovarian response, leading to higher doses of rFSH per oocyte and longer stimulation durations. The ESR1 variants correlated with a favorable response to COH, resulting in better embryo quality and higher likelihood of IVF success rates. Consequently, we extended the induction period and increased medication doses in subsequent IVF-ICSI cycles in 2023. In this cycle, FSH levels were slightly elevated at 5.2 mIU/mL on D2, suggesting a potential decline in ovarian reserve, with low LH levels indicating a slow ovarian response.^[7] However, elevated estradiol levels peaked at 1612.4 pg/mL after 13 days of stimulation, indicating a good follicular response. PRL was high at 54 ng/mL, which could disrupt normal ovulation. Overall, elevated estradiol and normal TSH during IVFICSI Cycle#1 created a favorable environment for follicular response.^[5] However, the pregnancy in IVF-ICSI cycle#1 was unsuccessful.

Role of ERA in personalized embryo transfer (pET)

Two frozen embryo transfer attempts were made using thawed embryos from previous IVF cycles. We performed embryo culture to employ blastocyst transfers, which are known to be viable and survive for 5 days and will improve implantation success rates. The first attempt of FET with two blastocysts (grades 4AA and 3AB) resulted in no pregnancy. Considering the woman’s hormonal fluctuations and low D2 LH levels, ERA was recommended to determine the optimal implantation window. ERA is a molecular diagnostic method that measures the gene expression profile of endometrial tissue.^[10] The ERA indicated that the pET should occur 144 ± 3 h after progesterone administration. The 2^nd attempt of FET of IVF-ICSI cycle#2 was carried out with two blastocysts, based on her personalized ERA results. β-hCG levels were measured 10–12 days post-blastocyst transfer, confirming a positive pregnancy at 1073 mIU/mL. Subsequent measurements showed satisfactory doubling rates of the beta hCG levels. An early pregnancy scan confirmed a single, live intrauterine gestation with good cardiac activity. The couple welcomed a healthy baby girl at full term in September, 2024.