Women undergoing in vitro fertilization have long worried that the procedure could raise their risk for breast cancer.

After all, the treatment requires temporarily increasing levels of certain sex hormones to five or 10 times the normal. Two of those hormones, estrogenand progesterone, can affect the course of certain kinds of breast cancer.

A series of studies over the past decade suggested that these former patients may have little to worry about. Experts remained cautious, however, because women who had undergone I.V.F. in the 1980s had not yet reachedmenopause by the time of the research.

But the largest, most comprehensive study to date, published Tuesday, provides further reassurance: It finds no increased risk among women who have undergone I.V.F.

“The main takeaway is there’s no evidence of an increased subsequent risk of breast cancer, at least in the first couple decades,” said Dr. Saundra S. Buys, an oncologist at the Huntsman Cancer Institute at the University of Utah, who was not involved in the new study.

The issue has nagged at specialists in reproductive medicine for some time. In 2008, a retrospective analysis of medical records, which the authors called “preliminary,” found a potential increase in breast cancer amongI.V.F. patients older than 40.

Another small study of participants at a treatment center in Israel reported an increased risk of breast cancer among women who start I.V.F. after 30.

Maddeningly, later findings went the other way, seeming to suggest the danger — if there was one — may be greater for younger women.

A study with roughly 21,000 participants, published in 2012, found that women in Western Australia who began I.V.F. at 24 or younger had an increased risk of breast cancer. No such link was found among women in their 30s or 40s.

In 2013, though, researchers published a meta-analysis of eight smaller studies tentatively suggesting that I.V.F. did not seem to raise breast cancer risk over all.

But it did not rule out the possibility that breast cancer might turn up in a bigger group of women tracked more closely for an even longer period. Experts also worried that infertility itself, not only its treatment, might somehow be linked to breast cancer.

Tuesday’s report, published in JAMA, goes a long way toward answering the lingering questions.

The huge study not only found no increased risk among women receiving I.V.F., but also found no greater risk among women who had various types of less intensive treatments to improve fertility.

More than 25,000 Dutch women, with an average age of 32.8 when they started treatment from 1980 to 1995, were followed for a median period of 21 years.

The researchers took into account an exhaustive list of factors linked to higher risk of cancer, including each woman’s age at the time she gave birth to her first child, her overall number of births and the number of I.V.F. attempts.

Because I.V.F. patients tend to have babies later in life than women who do not need assistance, “you have to take that into account,” said Alexandra van den Belt-Dusebout, the study’s first author and an epidemiologist at the Netherlands Cancer Institute in Amsterdam.

More than five million babies have been born worldwide through I.V.F. and other assisted reproduction.

Perhaps the study’s most surprising finding was that breast cancer risk was significantly lower among those women who underwent seven or more cycles of I.V.F., compared with those who received one or two cycles.

“That’s reassuring, because you would think if you did I.V.F. 10 times, your risk would be higher,” said Dr. Owen K. Davis, the president of the American Society for Reproductive Medicine.

The study also showed that women who responded poorly to ovarian stimulation in the first I.V.F. attempt also had decreased breast cancer risk.

Louise M. Stewart, a researcher at the Center for Population Health Research at Curtin University in Perth, Australia, speculated that the finding might explain why women who had I.V.F. at 24 or younger have an increased risk of breast cancer.

“Young women generally respond well to I.V.F. treatment,” said Dr. Stewart, who was the first author on the Australia study. She suggested the “increased risk we observed in young women may be related to their response to I.V.F. treatment.”

Mia Gaudet, the strategic director of breast and gynecologic cancer research at the American Cancer Society, applauded the study for adding a “significant amount of evidence that there is no link between I.V.F. and breast cancer.”

But she warned, “It’s still not conclusive.” For one thing, today’s protocols for I.V.F. differ slightly in the kinds of drugs given and for how long, the researchers noted.

The researchers have recruited an additional 10,000 Dutch women who had the latest I.V.F. regimen and 5,000 who received other fertility treatments. They will be tracking their health, as well.

Also, only 14 percent of participants had reached age 60, so this study cannot say much about postmenopausal breast cancer risk.

“We just may not be seeing breast cancer in these women yet,” Dr. Gaudet said.

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A version of this article appears in print on July 20, 2016, on page A15 of the New York edition with the headline: In Vitro Fertilization Is Found to Not Increase Chances of Breast Cancer. Order Reprints| Today's Paper|Subscribe